᪂つヨ㦂ἲᥦ᱌᭩             ᖹᡂ  ᖺ  ᭶  ᪥                                  1R 
ගᏳ඲ᛶホ౯ἲ 526 ࢔ࢵࢭ࢖
࡟㛵ࡍࡿᥦ᱌
 ᖹᡂ  ᖺ  ᭶  ᪥࡟ᮾிࠊᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ࡟࡚㛤ദࡉࢀࡓ᪂つヨ㦂ἲホ౯఍㆟㸦㏻⛠㸸-D&9$0 ホ౯఍㆟㸧࡟࠾࠸࡚௨ୗࡢᥦ᱌ࡀ࡞ࡉࢀࡓࠋ   ᥦ᱌ෆᐜ㸸 526㸦5HDFWLYH2[\JHQ6SHFLHV㸧࢔ࢵࢭ࢖ࡣࠊ㐺⏝㝈⏺࡟␃ពࡍࢀࡤࠊග໬Ꮫ཯ᛂ
ᛶ࡟㛵ࡋ࡚ഇ㝜ᛶࡢホ౯ࢆࡍࡿࡇ࡜ࡀ࡞࠸ࡇ࡜࠿ࡽࠊࡉࡽ࡞ࡿගᏳ඲ᛶホ౯ࡢせྰࡢุ᩿࡟฼⏝࡛ࡁࡿࠋ ᖺ࡟ᮏヨ㦂ἲࡣ་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ
࡚᥇ᢥࡉࢀࠊ᪤࡟⾜ᨻⓗ࡞ཷࡅධࢀࡀጞࡲࡗ࡚࠸ࡿࠋᮏヨ㦂ἲࢆගᏳ඲ᛶࡢホ౯ᡓ␎࡟⤌ࡳධࢀࡿࡇ࡜ࡣࠊ715837 ࡸࡑࢀ࡟⥆ࡃື≀ヨ㦂ࡢᐇ᪋ᩘࢆ๐ῶࡍࡿࡇ࡜
࡟ࡘ࡞ࡀࡿࠋ໬⢝ရ࣭་⸆㒊እရཎᩱࠊ㎰⸆ࠊࡑࡢ௚୍⯡໬Ꮫ≀㉁࡟ࡘ࠸࡚ࡢගᏳ඲ᛶホ౯ἲ࡜ࡋ࡚ࠊ⾜ᨻୖࡢ฼⏝ࡀᮇᚅ࡛ࡁࡿࠋ
ࡇࡢᥦ᱌᭩ࡣࠊ་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥࠊ526࢔ࢵࢭ࢖ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࠾ࡼࡧ㛵㐃ㄽᩥࢆࡶ࡜࡟ࠊගẘᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾࡲ࡜ࡵࡽࢀࡓᩥ᭩ࢆ⏝࠸࡚ࠊ-D&9$0ホ౯఍㆟ࡀホ౯࠾ࡼࡧ᳨ウࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽసᡂࡉࢀࡓࠋ ௨ୖࡢ⌮⏤࡟ࡼࡾࠊ⾜ᨻᙜᒁࡢᏳ඲ᛶホ౯᪉ἲ࡜ࡋ࡚ගᏳ඲ᛶホ౯ἲ 526 ࢔ࢵࢭ࢖ࡢ౑⏝ࢆᥦ᱌ࡍࡿࡶࡢ࡛࠶ࡿࠋ ኱㔝Ὀ㞝                      すᕝ⛅ెᕝ⛅ెJaCVAM ホ౯఍㆟ ㆟㛗               JaCVAM 㐠Ⴀጤဨ఍ ጤဨ㛗i
-D&9$0 ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤ሯᑦᩥ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧▼஭㞝஧㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ᒾ℩⿱⨾Ꮚ㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧㔠Ꮚ࿴ᘯ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧⠛⏣࿴ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧㇂ᕝᾈᏊ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᒣ⏣㝯ᚿ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧ྜྷ⏣Ṋ⨾㸦᪥ᮏẘᛶᏛ఍㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧                  ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥ii

-D&9$0 㐠Ⴀጤဨ఍すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧㸸ጤဨ㛗ᕝす ᚭ  㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧ᑠᕝஂ⨾Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ຍ⸨ ⠜

㸦ᅜ❧ឤᰁ⑕◊✲ᡤ㸧Ⳣ㔝 ⣧

㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊㸧⠛⏣࿴ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧㛵㔝♸Ꮚ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⸆⌮㒊㸧㧗ᮌ⠜ஓ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊 ື≀⟶⌮ᐊ㸧᮰㔝ṇ᫂㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧୰ᮧ㧗ᩄ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧᪥⏣ ඘

㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧ᗈ℩᫂ᙪ  㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶண
ホ౯㒊㸧
ᮏ㛫ṇ඘

㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ኚ␗㑇ఏ㒊㸧⨾ୖ᠇୍㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧୕⃝ 㤾㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁᑂᰝ⟶⌮ㄢ㸧ᑠᓥ ⫕㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶணホ౯㒊 ➨஧ᐊ㸧㸸஦ົᒁ

iii

iv

JaCVAM statement on the Reactive Oxygen Species (ROS) assay forphotosafety assessment
At a meeting held on 17 December 2015 at the National Institute of Health Sciences (NIHS)
in Tokyo, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:
Proposal: Provided that proper consideration is given to the limits of applicability, theReactive Oxygen Species (ROS) assay is a useful means of determining the needfor additional photosafety assessment, because it does not produce false negatives
regarding photochemical reactivity. Guidelines for the photosafety assessment ofdrugs using the ROS assay were approved in 2014 and are increasingly accepted
for regulatory use.
The incorporation of the ROS assay into photosafety assessment strategies can be
expected to help reduce the need for 3T3 NRU PT as well as follow-up testing
using animals. We look forward its use in a regulatory context for the photosafetyassessment of ingredients used in cosmetics and quasi-drugs, agriculturalchemicals, and other chemical substances.
This statement was prepared following a review of guidelines for the photosafety assessmentof drugs, the ROS Assay Validation Report, and related documentation and using materials
prepared by the Phototoxicity Testing JaCVAM Editorial Committee to acknowledge that theresults of a review and study by the JaCVAM Regulatory Acceptance Board haveacknowledged the usefulness of this assay.
Based on the above, we propose the ROS assay as a means for photosafety assessment insafety assessments by regulatory agencies.

Yasuo Ohno
Chairperson
JaCVAM Regulatory Acceptance Board

Akiyoshi Nishikawa
Chairperson
JaCVAM Steering Committee

20 January 2016v

The JaCVAM Regulatory Acceptance Board was established by the JaCVAM Steering
Committee, and is composed of nominees from the industry and academia.
This statement was endorsed by the following members of the JaCVAM Regulatory
Acceptance Board:

Mr. Yasuo Ohno (nominee by JaCVAM Steering Committee) : Chairperson
Mr. Naofumi Iizuka (Pharmaceuticals and Medical Devices Agency)
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Yuji Ishii (NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Kazuhiro Kaneko (Japan Chemical Industry Association)
Mr. Eiji Maki (Japanese Society of Immunotoxicology)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Akiyoshi Nishikawa (NIHS)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Ms. Koko Tanigawa (Japanese Society for Alternatives to Animal Experiments)
Mr. Takashi Yamada (National Institute of Technology and Evaluation)
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact Dermatitis)
Mr. Takemi Yoshida (Japanese Society of Toxicology)
Mr. Isao Yoshimura (nominee by Chairperson)
Term: From 1st April 2014 to 31st March 2016vi

This statement was endorsed by the following members of the JaCVAM steering Committee
after receiving the report from JaCVAM Regulatory Acceptance Board:

Mr. Akiyoshi Nishikawa (BSRC, NIHS): Chairperson
Mr. Toru Kawanishi (NIHS)
Mr. Mitsuru Hida (Ministry of Health, Labour and Welfare)
Mr. Akihiko Hirose (Division of Risk Assessment, BSRC, NIHS)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, BSRC, NIHS)
Mr. Jun Kanno (Division of Cellular and Molecular Toxicology, BSRC, NIHS)
Mr. Atsushi Kato (National Institute of Infectious Diseases)
Mr. Kenichi Mikami (Ministry of Health, Labour and Welfare)
Mr. Kaoru Misawa (Ministry of Health, Labour and Welfare)
Mr. Takatoshi Nakamura (Pharmaceutical & Medical Devices Agency)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Ms. Yuko Sekino (Division of Pharmacology, BSRC, NIHS)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Mr. Atsuya Takagi (Animal Management Section of the Division of Cellular andMolecular Toxicology, BSRC, NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)Mr. Hajime Kojima (Section for the Evaluation of Novel Methods, Division of Risk
Assessment, BSRC, NIHS): Secretary

vii

viii

ගᏳ඲ᛶホ౯ἲ㸸ROS ࢔ࢵࢭ࢖┠  ḟ
ホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------ 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------ 7་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ࡟ࡘ࠸࡚----------------------------------------- 53
Peer Review Panel Evaluation of the Reactive Oxygen Species (ROS)
Photosafety Assay ----------------------------------------------------------------------------- 71
REACTIVE OXYGEN SPECIES (ROS) ASSAY TO EXAMINEPHOTOREACTIVITY OF CHEMICALS---------------------------------------------------87ix

x

ホ౯఍㆟ሗ࿌᭩
Reactive oxygen species (ROS)࢔ࢵࢭ࢖

ᖹᡂ 27 ᖺ㸦2015 ᖺ㸧11 ᭶ 30 ᪥

JaCVAM ホ౯఍㆟

11

JaCVAM ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤ሯᑦᩥ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼஭㞝஧㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧㔠Ꮚ࿴ᘯ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧⠛⏣࿴ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧㇂ᕝᾈᏊ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᒣ⏣㝯ᚿ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧ྜྷ⏣Ṋ⨾㸦᪥ᮏẘᛶᏛ఍㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧                ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥

22

Reactive oxygen species (ROS)࢔ࢵࢭ࢖ࡣࠊ໬Ꮫ≀㉁ࡢග཯ᛂᛶࢆ᳨ฟࡍࡿヨ㦂ἲ࡛࠶ࡿࠋග᭚㟢࡟ࡼࡾ⏕ࡌࡿ໬Ꮫ≀㉁ࡢẘᛶࡢከࡃࡣࠊ໬Ꮫ≀㉁ࡢග໬Ꮫ཯ᛂ࡟క࠺⣽⬊ࡸ⤌⧊ࡢ㓟໬ⓗയᐖࡀⓎ⌧ᶵᗎ࡜ࡉࢀࠊගບ㉳ࡉࢀࡓ໬Ꮫ≀㉁ࡣ୍㔜㡯㓟⣲ࡸࢫ࣮ࣃ࣮࢜࢟ࢩࢻ
࢔ࢽ࢜ࣥ➼ࡢ ROS ࢆ⏘⏕ࡍࡿࠋROS ࢔ࢵࢭ࢖ࡣࠊ໬Ꮫ≀㉁࡟ᨃఝኴ㝧ගࢆ↷ᑕࡋࡓ࡜ࡁࡢ
ROS ࡢ⏘⏕ࡢ᭷↓ࢆㄪ࡭ࡿࡇ࡜࡟ࡼࡾࠊග཯ᛂᛶࢆணࡍࡿࠋ

ROS ࢔ࢵࢭ࢖ࡣࠊJaCVAM ༠ຊࡢୗ࡛ከ᪋タࣂࣜࢹ࣮ࢩࣙࣥࡀᐇ᪋ࡉࢀ 1)ࠊOECD ࡀᐃࡵࡿᅜ㝿ⓗᇶ‽(2005 ᖺ) 2)࡟ᚑࡗ࡚ࠊᑓ㛛ᐙ࠿ࡽ࡞ࡿ⊂❧ࡋࡓ➨୕⪅ホ౯ጤဨ఍࡟ࡼࡾࠊ⛉Ꮫⓗ࡞ホ౯ࡀ⾜ࢃࢀࡓ 3)ࠋJaCVAM ホ౯఍㆟ࡣࠊࡇࢀࢆࡶ࡜࡟ࡋ࡚ JaCVAM ගẘᛶ㈨ᩱ⦅
⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓ ROS ࢔ࢵࢭ࢖ホ౯ሗ࿌᭩㸦2015 ᖺ㸵᭶ 8 ᪥㸧4)ࢆ⏝࠸࡚ࠊᮏヨ㦂ἲࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 Reactive oxygen species (ROS)࢔ࢵࢭ࢖
௦ ᭰ ࡍ ࡿ ᑐ ㇟ ẘ ᛶ ヨ 㦂 㸸  In vivo ࡢ ග ẘ ᛶ ヨ 㦂 5) ࠾ ࡼ ࡧ Neutral Red UptakePhototoxicity Test㸦௨ୗࠊ3T3 NRU PT ࡜␎ࡍ )6)ヨ㦂ヨ㦂ἲࡢᴫ␎㸸  ᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁࡟ᨃఝኴ㝧ගࢆ↷ᑕࡋࠊROS ୰ࡢ୍㔜㡯㓟⣲࡜ࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥࡢ 2 ✀ࡢ⏘⏕ࡢ᭷↓ࢆㄪ࡭ࡿヨ㦂࡛࠶ࡿࠋ୍㔜㡯㓟⣲ࡣ pnitrosodimethylaniline ࡢኚⰍࢆᣦᶆ࡜ࡋ࡚ࡑࡢ⏘⏕ࢆホ౯ࡍࡿࠋࡍ࡞ࢃࡕ㸪⿕㦂≀㉁ࡀග↷ᑕࢆཷࡅ࡚ບ㉳໬ྜ≀࡜࡞ࡾࠊࡇࢀ࠿ࡽ⏘⏕ࡉࢀࡓ୍㔜㡯㓟⣲ࡀ཯ᛂᾮ୰ࡢ imidazole ࡜཯ᛂࡋ࡚཯ᛂ୰㛫యࢆᙧᡂࡍࡿࠋࡇࢀࡀ p-nitrosodimethylaniline ࢆ㓟໬ࡍࡿࡇ࡜࡛ኚⰍࢆ
ࡶࡓࡽࡋ㸪440 nm ࡢ྾ග್ࡀῶᑡࡍࡿࠋࡇࡢ྾ග್ࡢῶᑡࢆ୍㔜㡯㓟⣲ࡢ⏕ᡂ࡜ࡋ࡚ᐃࡍࡿࠋsinglet oxygen + imidazole Ѝ [peroxide intermediate] Ѝ oxidized imidazole[peroxide intermediate] + p-nitrosodimethylaniline Ѝ oxidized p-nitrosodimethylaniline +productsࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥࡢ⏘⏕ࡣࠊnitroblue tetrazolium (NBT) ࡢ㑏ඖࢆᣦᶆ࡜ࡍࡿࠋບ
㉳໬ྜ≀࠿ࡽ⏘⏕ࡉࢀࡿ ࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥࡣ୍㟁Ꮚ㌿⛣཯ᛂ࡟ࡼࡾ NBT ࢆ㑏ඖࡋࠊ⏕ᡂࡉࢀࡿ monoformazan ࡣ 560 nm ࡟ UV ྾཰ࢆ᭷ࡍࡿࠋࡇࡢ྾ග್ࡢቑຍࢆࢫ࣮
ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥࡢ⏕ᡂࡢᣦᶆ࡜ࡋ࡚

ᐃࡍࡿࠋsuperoxide + NBT ĺ O2 + monoformazan (NBT+)
㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶ JaCVAM ༠ຊࡢୗ࡛ᐇ᪋ࡉࢀࡓከ᪋タࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢ⤖ᯝࡀࠊOECD ࡀᐃࡵࡓᅜ㝿ⓗᇶ‽㸦2005 ᖺ)2㸧࡟ᚑࡗ࡚ࠊࣂࣜࢹ࣮ࢩࣙࣥᐇ᪋ᶵ㛵࡜ࡣ⊂❧ࡋࡓᑓ㛛ᐙ࠿ࡽ࡞ࡿ➨33୕⪅ホ౯ጤဨ఍࡟࡚⛉Ꮫⓗ࡞ホ౯ࡸ᳨ドࡀ⾜ࢃࢀࠊࡑࡢ⤖ᯝࢆࡶ࡜࡟ JaCVAM ගẘᛶ㈨ᩱ
⦅⧩ጤဨ఍ࡀሗ࿌᭩࡜ࡋ࡚ࡲ࡜ࡵࡓࡶࡢࢆホ౯㈨ᩱ࡜ࡋࡓࠋ
໬Ꮫ≀㉁࡟ኴ㝧ගࢆ↷ᑕࡋࡓሙྜࠊᚓࡽࢀࡓບ㉳࢚ࢿࣝࢠ࣮ࡣ⏕యෆ≀㉁ࡸ㓟⣲ศᏊ࡟㌿⛣ࡋࡓࡾࠊ࠶ࡿ࠸ࡣ໬ྜ≀ࡑࡢࡶࡢࡀ⏕యෆ≀㉁࡜⤖ྜࡋ࡚ග௜ຍ≀ࢆᙧᡂࡍࡿࠋࡲࡓࠊ
㓟⣲ศᏊࡣບ㉳࢚ࢿࣝࢠ࣮ࡢ࢔ࢡࢭࣉࢱ࣮࡜࡞ࡾࠊࡑࢀ࡟క࠸⏘⏕ࡉࢀࡓ୍㔜㡯㓟⣲ࡸ ࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥ➼ࡢ ROS ࡟ࡼࡿ⏕యෆ≀㉁ࡢ㓟໬཯ᛂࡀ⸆๣ᛶග⥺㐣ᩄ⑕ࡢⓎ⑕ཎᅉ࡜ࡋ࡚⪃࠼ࡽࢀ࡚࠸ࡿ 7)ࠋROS ࡣࠊගẘᛶ≀㉁ࡢࡳ࡛࡞ࡃග࢔ࣞࣝࢠ࣮ࢆ♧ࡍ≀㉁࡛ࡶ
Ⓨ⏕ࡋࠊගࢱࣥࣃࢡ⤖ྜࡲࡓࡣගศゎ࡟ࡘ࡞ࡀࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ 8)ࠋ௨ୖࡢࡼ࠺࡟ࠊᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁࡟ᨃఝኴ㝧ගࢆ↷ᑕࡋࠊ⏘⏕ࡍࡿ୍㔜㡯㓟⣲࡜ࢫ࣮ࣃ࣮࢜࢟ࢩࢻ࢔ࢽ࢜ࣥ
ࡢ᭷↓ࢆㄪ࡭ࡿග໬Ꮫⓗ཯ᛂᛶヨ㦂࡛࠶ࡿࡇ࡜࠿ࡽࠊගᏳ඲ᛶࡢホ౯ἲ࡜ࡋ࡚ཎ⌮ⓗ࡟ጇᙜᛶࡀ࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ
㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
ᮏヨ㦂ἲࡣࠊື≀࠶ࡿ࠸ࡣື≀⏤᮶ࡢ⏕యヨᩱࢆ౑⏝ࡋ࡞࠸ࡇ࡜࠿ࡽࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠸ࡿࠋࡲࡓ໬Ꮫ≀㉁ࡢග໬Ꮫⓗ཯ᛂࢆ㎿㏿࠿ࡘ⡆౽࡟ᐃ࡛ࡁࠊከ᳨యฎ⌮ࡀྍ⬟࡛࠶ࡿࠋࡉࡽ࡟ࢥࢫࢺ㠃࡟࠾࠸࡚ࡶඃࢀ࡚࠸ࡿࠋከ᪋タࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ࠊ㞴⁐ᛶࡢࡓࡵホ౯࡛ࡁ࡞࠿ࡗࡓ⿕㦂≀㉁ࢆ㝖ࡃ࡜ࠊᮏヨ㦂ἲ࡟ࡼࡿගẘᛶ㝧ᛶ≀㉁ࡢ᳨ฟ⋡ࡣ 100%࡛࠶ࡾࠊഇ㝜ᛶ⤖ᯝࡀ࡞࠸ࡇ࡜࠿ࡽࠊගẘᛶࡢࣁࢨ࣮ࢻホ౯࡟᭷⏝࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋࡉࡽ࡟ࠊ་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ
9)࡟࠾࠸࡚ࠊᮏヨ㦂ἲࡣึᮇホ౯㡯┠ࡢࡦ࡜ࡘ࡜ࡋ࡚グ㍕ࡉࢀࠊᮏヨ㦂ἲ࡛㝜ᛶ࡜ุᐃࡉࢀ

ࡓ㛤Ⓨ୰ࡢ་⸆ရ࡟ࠊࡉࡽ࡞ࡿගẘᛶヨ㦂ࢆ⾜࠺ᚲせࡣ࡞࠸࡜ࡉࢀ࡚࠸ࡿࠋࡑࡢࡓࡵࠊᮏヨ㦂ἲࢆගᏳ඲ᛶホ౯࡟ᑟධࡍࡿࡇ࡜࡟ࡼࡾࠊ௦᭰ࡍࡿගẘᛶヨ㦂ᩘࡢ๐ῶ࡟ࡘ࡞ࡀࡿࡇ࡜
ࡀᮇᚅ࡛ࡁࡿࠋ ḟ࡟ࠊ⣽⬊ࡸື≀ࢆ฼⏝ࡍࡿගᏳ඲ᛶホ౯⣔࡛ࡣࠊࡑࢀ⮬㌟ࡢẘᛶࡀᙉ࠸ UVB ࢆග※࡜ࡋ࡚౑⏝ࡍࡿࡇ࡜ࡀ㞴ࡋࡃࠊUVB 㡿ᇦ࡟ࡋ࠿྾཰ࢆ᭷ࡉ࡞࠸໬ྜ≀࡟ᑐࡋ࡚㐺⏝ࡀᅔ㞴࡞ሙྜࡀ࠶ࡿࡀࠊᮏヨ㦂ἲࡣ⿕㦂≀㉁⮬యࡢ≀ᛶࡢホ౯⣔࡛࠶ࡿࡓࡵ࡟ UVB ࡢ↷ᑕࡶྍ⬟࡛࠶ࡿࠋࡲࡓࠊ⏕యヨᩱࢆᚲせ࡜ࡋ࡞࠸ࢩࢫࢸ࣒࡛࠶ࡿࡓࡵࠊࡑࡢ෌⌧ᛶࡶ㧗࠸ࠋ୍᪉ࠊ㐺⏝㝈⏺࡜ࡋ࡚ࠊ཯ᛂᾮ୰࡛ᯒฟࡍࡿ⁐ゎᛶࡢప࠸≀㉁ࡸ⁐ゎ᫬࡟╔Ⰽࡀㄆࡵࡽࢀࡿ໬Ꮫ≀㉁ࡣホ౯࡛ࡁ࡞࠸ࠋࡲࡓ⿕㦂≀㉁ࡣࣔࣝ⃰ᗘ༢఩࡛ヨ㦂ࡍࡿࡼ࠺ࣉࣟࢺࢥࣝࡀタᐃࡉࢀ࡚࠸ࡿࡓࡵࠊᢳฟ≀ࡸΰྜ≀ࡢࡼ࠺࡟ศᏊ㔞ࡀ୙࡛᫂ࣔࣝ⃰ᗘࢆ⟬ฟ࡛ࡁ࡞࠸⿕㦂≀㉁ࡣホ౯࡛ࡁ࡞࠸ࠋࡇࡢࡼ࠺࡞ヨ㦂᪉ἲࡢㄢ㢟࡟ᑐࡋࠊྛࠎࠊ⏺㠃άᛶ๣ࡸ࢔ࣝࣈ࣑ࣥࢆຍ࠼ࡓ᪉
ἲ 10-12)ࠊᣦ♧⸆ࢆῧຍࡋ࡞࠸ヨ㦂ࢥࣥࢺ࣮ࣟࣝ⁐ᾮࡢタᐃ 13)ࠊPg/mL ༢఩࡛ヨ㦂ࡍࡿ᪉ἲ
14)➼ࡢᑐᛂἲࡀ᳨ウࡉࢀ࡚࠾ࡾࠊᢳฟ≀ࡸΰྜ≀➼ࡢከ࠸໬⢝ရཎᩱࢆࡣࡌࡵࠊ୍⯡໬Ꮫ≀㉁࡞ࡽࡧ࡟㎰⸆࡟ࡘ࠸࡚ࡢගᏳ඲ᛶホ౯ἲ࡜ࡋ࡚᭷⏝ᛶࡀᮇᚅ࡛ࡁࡿࡀࠊࡲࡔホ౯᪉ἲ࡜ࡋ࡚☜❧ࡣࡉࢀ࡚࠸࡞࠸ࠋ
ࡲࡓࠊගᏳ඲ᛶࡢ㐺⏝⠊ᅖ࡜ࡋ࡚ගẘᛶ࠾ࡼࡧග࢔ࣞࣝࢠ࣮ࡣ࡜ࡶ࡟Ⓨ⌧ᶵᗎࡢୖὶ࡟
44ග໬Ꮫ཯ᛂࡀᏑᅾࡍࡿࡇ࡜ࠊࡉࡽ࡟ࠊග࢔ࣞࣝࢠ࣮≀㉁ࡶᮏヨ㦂ἲ᳨࡛ฟ࡛ࡁࡿ࡜ࡢ▱ぢࡀᚓࡽࢀ࡚ࡁࡓࡇ࡜࠿ࡽ 3)ࠊ㐺ษ࡟ᐇ᪋ࡉࢀࡓ ROS ࢔ࢵࢭ࢖࡛㝜ᛶ࡛࠶ࢀࡤගẘᛶࠊග࢔ࣞ
ࣝࢠ࣮ࢆ♧ࡉ࡞࠸࡜ุ᩿ࡉࢀࡿࡀࠊ㝧ᛶࡢሙྜࡣࠊู㏵ホ౯ࡉࢀࡿ࡭ࡁ࡛࠶ࡿࠋ
㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධࢀ࠾ࡼࡧ⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸 ᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁ࡢග໬Ꮫⓗ≉ᛶࢆᣦᶆ࡜ࡋࡓ⡆౽࠿ࡘື≀࠾ࡼࡧື≀⏤᮶ヨᩱࢆ
⏝࠸࡞࠸ヨ㦂ἲ࡛࠶ࡾࠊᨃఝኴ㝧ග↷ᑕ⿦⨨(solar simulator)ࠊUVAᐃ⿦⨨ࠊ࣐࢖ࢡࣟ

ࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ࡀ฼⏝࡛ࡁࡿ᪋タ࡛࠶ࢀࡤᐜ᫆࡟ᐇ᪋࡛ࡁࡿヨ㦂࡛࠶ࡿࠋᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁ࡢග໬Ꮫⓗ≉ᛶࢆᣦᶆ࡜ࡋࡓගẘᛶ࠾ࡼࡧග࢔ࣞࣝࢠ࣮ࡢⓎ⌧ᶵᗎ࡟࠾ࡅࡿୖὶࡢ㔜せ࡞࢖࣋ࣥࢺࢆ᳨ฟࡋ࡚࠾ࡾࠊ໬Ꮫ≀㉁ࡢගᏳ඲ᛶࢆ⪃࠼ࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼ࡿࡇ
࡜࠿ࡽࠊᮏヨ㦂ἲࡢ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࠋ࡞࠾ࠊᮏヨ㦂ἲࡢᐇ᪋࡟ᙜࡓࡗ࡚ࡣࠊ⿕㦂≀㉁ࡢ≉ᛶ࡜ヨ㦂ἲࡢ㐺⏝㝈⏺ࢆぢᴟࡵࠊᚓࡽࢀࡓ⤖ᯝࡢゎ㔘࡟ὀពࢆᡶ࠺ᚲせࡀ࠶ࡿࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸 ᮏヨ㦂ἲࡣࠊ㐺⏝㝈⏺࡟␃ពࡍࢀࡤࠊග໬Ꮫ཯ᛂᛶ࡟㛵ࡋ࡚ഇ㝜ᛶࡢホ౯ࢆࡍࡿࡇ࡜ࡀ࡞
࠸ࡇ࡜࠿ࡽࠊࡉࡽ࡞ࡿගᏳ඲ᛶホ౯ࡢせྰࡢุ᩿࡟฼⏝࡛ࡁࡿࠋ2014 ᖺ࡟ᮏヨ㦂ἲࡣ་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚᥇ᢥࡉࢀࠊ᪤࡟⾜ᨻⓗ࡞ཷࡅධࢀࡀጞࡲࡗ࡚࠸ࡿࠋᮏ
ヨ㦂ἲࢆගᏳ඲ᛶࡢホ౯ᡓ␎࡟⤌ࡳධࢀࡿࡇ࡜ࡣࠊ3T3 NRU PT ࡸࡑࢀ࡟⥆ࡃື≀ヨ㦂ࡢᐇ᪋ᩘࢆ๐ῶࡍࡿࡇ࡜࡟ࡘ࡞ࡀࡿࠋ໬⢝ရ࣭་⸆㒊እရཎᩱࠊ㎰⸆ࠊࡑࡢ௚୍⯡໬Ꮫ≀㉁࡟
ࡘ࠸࡚ࡢගᏳ඲ᛶホ౯ἲ࡜ࡋ࡚ࠊ⾜ᨻୖࡢ฼⏝ࡀᮇᚅ࡛ࡁࡿࠋᘬ⏝ᩥ⊩1) Onoue S, Hosoi K., Toda T., Takagi H., Osaki N., Matsumoto Y., Kawakami S., Wakuri
S., Iwase Y., Yamamoto T., Nakamura K., Ohno Y., Kojima H. (2014) Intra-/interlaboratory validation study on reactive oxygen species assay for chemical photosafety
evaluation using two different solar simulators. Toxicol. In Vitro, 28(4), 515-523.
2) OECD GUIDANCE DOCUMENT ON THE VALIDATION AND INTERNATIONALACCEPTANCE OF NEW OR UPDATED TEST METHODS FOR HAZARD ASSESSMENT
No.34 (2005), OECD Series on Testing and Assessment: Testing for Human Health JaCVAM
Executive Summary of “Peer Review Panel Evaluation of the Reactive Oxygen
Species (ROS) Photosafety Assay” (16 October 2013)
3) JaCVAM ගẘᛶ㈨ᩱ⦅⧩ጤဨ఍㸸ROS ࢔ࢵࢭ࢖ホ౯ሗ࿌᭩(2015 ᖺ㸵᭶ 8 ᪥)4) Morikawa F., Nakayama Y., Fukuda M., Yokoyama Y., Nagura T., Ishihara M., Toda
K. (1974) Techniques for evaluation of phototoxicity and photoallergy in laboratory
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Ed. by Fitzpatrick, T.B. University ofTokyo Press, Tokyo, pp.529-557.
5) OECD (2004) Test Guideline 432. In Vitro 3T3 NRU phototoxicity test.
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7) Tokura Y. (2009) Photoallergy. Expert Rev. Dermatol., 4(3), 263-270.
8) ཌ⏕ປാ┬㸦2014㸧་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ࡟ࡘ࠸࡚㸦⸆㣗ᑂᰝⓎ 0521 ➨1 ྕ㸪ᖹᡂ 26 ᖺ 5 ᭶ 21 ᪥㸧9) Onoue S., Yamauchi Y., Kojima T., Igarashi N., Tsuda Y. (2008) Analytical studies onphotochemical behavior of phototoxic substances; Effect of detergent additives on
singlet oxygen generation. Pharm. Res., 25, 861–868.
10) Onoue S, Kato M, Yamada S (2014) Development of an albuminous reactive oxygen
species assay for photosafety evaluation under experimental biomimetic conditions,
J. Appl. Toxicol., 34: 158–65.
11) Seto Y, Kato M, Yamada S, Onoue S. (2013) Development of micellar reactive oxygen
species assay for photosafety evaluation of poorly water-soluble chemicals. Toxicol.
in Vitro, 27: 1838–46.
12) Onoue S, Suzuki G, Kato M, Hirota M, Nishida H, Kitagaki M, Kouzuki H, Shizuo
Yamada S. (2013) Non-animal photosafety assessment approaches for cosmetics
based on the photochemical and photobiochemical properties, Toxicol. in Vitro, 27:
2316–24
13) Nishida H, Hirota M, Seto Y, Suzuki G, Kato M, Kitagaki M, Sugiyama M, Kouzuki
H, Onoue S.(2015) Non-animal photosafety screening for complex cosmetic
ingredients with photochemical and photobiochemical assessment tools., Regul.
Toxicol. Pharmacol., 72(3) 578-585.

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ROS ࢔ࢵࢭ࢖ホ౯ሗ࿌᭩2015 ᖺ 7 ᭶ 8 ᪥

JaCVAM ගẘᛶ㈨ᩱ⦅⧩ጤဨ఍7ጤဨ㛗ᇼ஭ 㑳ኵ

㸦ࣇ࢓࢖ࢨ࣮ᰴᘧ఍♫㸧ጤ ဨᑿୖ ㄔⰋ

㸦㟼ᒸ┴❧኱Ꮫ㸧㔠Ꮚ ࿴ᘯ

㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧ୖ᭶ ⿱୍

㸦ᰴᘧ఍♫ ㈨⏕ᇽ ⏣୰ ᠇✑㸦㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮㸧➜ᮌ ಟ

㸦་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧⣽஭ ୍ᘯ

㸦ཧኳ〇⸆ᰴᘧ఍♫㸧

2
8

┠ḟ
i.
␎ㄒ ...................................................................................................................... 4ii.
せ᪨ ...................................................................................................................... 5
1.
ヨ㦂᪉ἲࡢ⛉Ꮫⓗ⫼ᬒ࣭᰿ᣐ࠾ࡼࡧつไୖࡢ఩⨨࡙ࡅ ....................................... 6
1.1⛉Ꮫⓗ⫼ᬒ............................................................................................................ 6
1.2⛉Ꮫⓗ᰿ᣐ............................................................................................................ 7
1.3
つไୖࡢ఩⨨࡙ࡅ ................................................................................................. 92ヨ㦂ࡢࣉࣟࢺࢥࣝ࠾ࡼࡧุᐃᇶ‽ ..................................................................... 11
2.1
ᐃཎ⌮ ............................................................................................................. 11
2.2ᮦᩱ࠾ࡼࡧヨ⸆ .................................................................................................. 11
2.3⿕㦂≀㉁ ............................................................................................................. 12
2.4
㝧ᛶ࡞ࡽࡧ࡟㝜ᛶᑐ↷≀㉁ ................................................................................ 13
2.5
ჾල࣭ᶵჾ.......................................................................................................... 13
2.6
ヨ㦂᪉ἲ࡞ࡽࡧ࡟ࢹ࣮ࢱゎᯒ............................................................................. 14
2.7
ࢹ࣮ࢱ᥇⏝᮲௳ .................................................................................................. 17
2.8
㝧ᛶᇶ‽ ............................................................................................................. 183
ヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥ .................................................................................... 18
3.1⏝࠸ࡽࢀࡓ≀㉁࡜ࡑࡢጇᙜᛶ............................................................................. 18
3.2ࣂࣜࢹ࣮ࢩࣙࣥ⤖ᯝࡢホ౯ ................................................................................ 214
ヨ㦂᪉ἲ࡟㛵ࡍࡿ௚ࡢ⛉Ꮫⓗ⪃ᐹ ..................................................................... 25
4.1ග࡟୙Ᏻᐃ࡞≀㉁࡬ࡢ㐺⏝ ................................................................................ 25
4.2
㞴⁐ᛶ≀㉁࡬ࡢᑐᛂ ........................................................................................... 25
4.3
ΰྜ≀࠾ࡼࡧศᏊ㔞୙᫂ヨᩱ࡬ࡢᑐᛂ .............................................................. 26
4.4ග࢔ࣞࣝࢠ࣮ᛶண ࡬ࡢ฼⏝............................................................................. 28
5
3Rs ࡬ࡢ㛵୚㸦ື≀⚟♴㠃࠿ࡽࡢጇᙜᛶ㸧 ....................................................... 296ヨ㦂᪉ἲࡢ᭷⏝ᛶ࡜ၥ㢟Ⅼ ................................................................................ 29
7⤖ㄽ .................................................................................................................... 308⏝ㄒࡢゎㄝ.......................................................................................................... 319ཧ⪃ᩥ⊩ ............................................................................................................. 31
10
Appendix............................................................................................................. 34
Appendix 1 Executive Summary of “Peer Review Panel Evaluation of the Reactive Oxygen
Species (ROS) Photosafety Assay” (16 October 2013) ..................................... 34
Appendix 2 List of reasons for chemical selection .................................................................. 36
Appendix 3 Test chemicals for the Phase 1 study .................................................................. 38
Appendix 4 List of reasons for chemical selection .................................................................. 39
Appendix 5 Test chemicals for Phase 2 study and code list................................................... 45





3
9

i.

␎ㄒ
3T3 NRU-PT:

3T3 neutral red uptake phototoxicity test

CDER:
CPMP:

Center for Drug Evaluation and Research㸦་⸆ရホ౯◊✲ࢭࣥࢱ࣮㸧
Committee for Proprietary Medicinal Products㸦Ḣᕞ་⸆ရጤဨ఍㸧

DMSO:
EMEA:

Dimethyl sulfoxide
European Agency for the Evaluation of Medicinal Products㸦Ḣᕞ་⸆ရᑂᰝᗇ㸪2004 ᖺࡲ࡛౑ࢃࢀ࡚࠸ࡓ࿧⛠㸧

EMA:
EWG:

European Medicines Agency㸦Ḣᕞ་⸆ရᗇ㸧
Expert Working Group㸦ᑓ㛛ᐙసᴗ㒊఍㸧

FDA

Food and Drug Administration㸦⡿ᅜ㣗ရ་⸆ရᒁ㸧

IC:
ICH:

Internal conversion㸦ෆ㒊㌿⛣㸧International Conference on Harmonization of Technical

ISC:

Requirements for Registration of Pharmaceuticals for Human Use㸦᪥⡿ EU ་⸆ရつไㄪ࿴ᅜ㝿఍㆟㸧
Intersystem crossing㸦㡯㛫஺ᕪ㸧

NaPB:
NBT:
NBT+:
OECD:
RNO:
ROS:
UVA:
UVB:
UVC:
UV/VIS:

Sodium Phosphate Buffer
Nitroblue tetrazolium
Monoformazan
Organisation for Economic Co-operation and Development㸦⤒῭༠ຊ㛤Ⓨᶵᵓ㸧p-nitrosodimethyl aniline
Reactive Oxygen Species, including superoxide anion (SA) andsinglet oxygen (SO).
Ultraviolet light A (wavelengths between 320 and 400 nm).
Ultraviolet light B (wavelengths between 290 and 320 nm).
Ultraviolet light C (wavelengths between 190 and 290 nm).
Ultraviolet and visible light

4
10

LL

せ᪨
Reactive oxygen species (ROS) ࢔ࢵࢭ࢖ࡣ㸪໬Ꮫ≀㉁ࡢග໬Ꮫⓗ≉ᛶࢆᣦᶆ࡜
ࡋࡓ⡆౽࡛ከ᳨యฎ⌮ྍ⬟࡞ගᏳ඲ᛶホ౯ἲ࡜ࡋ࡚ᥦ᱌ࡉࢀ㸪໬Ꮫ≀㉁࡟ᨃఝ
ኴ㝧ගࢆ↷ᑕࡋ㸪singlet oxygen ࡜ super oxide anion ࡢ⏘⏕ࡢ᭷↓ࢆㄪ࡭ࡿග཯
ᛂᛶヨ㦂ࡢ୍ࡘ࡜ࡋ࡚㸪ග⥺㐣ᩄ⑕ㄏⓎࡢྍ⬟ᛶࢆண ࡋᚓࡿࡇ࡜ࡀ᫂ࡽ࠿࡟
ࡉࢀ࡚࠸ࡿ㸬ࡇࡢ ROS ࢔ࢵࢭ࢖ࡣ 2014 ᖺ࡟ step 5 ࡟฿㐩ࡋࡓ ICH S10 ࢞࢖ࢻࣛ࢖ࣥ࡟࠾࠸࡚ࡶ࡜ࡾୖࡆࡽࢀ࡚࠾ࡾ
ࠕࢹ࣮ࢱ࠿ࡽࡣ㸪ࡇࡢヨ㦂ἲࡣ in vivo ࡟࠾ࡅࡿ┤᥋ⓗ࡞ගẘᛶ≀㉁ࢆணぢࡍࡿୖ࡛ࡢឤᗘࡀ㧗࠸ࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿࠖ
࡜グ㏙ࡉࢀ࡚࠸ࡿ㸬
ROS ࢔ࢵࢭ࢖ࡢ᭷⏝ᛶホ౯࡜ࡋ࡚㸪JaCVAM ᣦᑟୗ࡛ࡢከ᪋タࣂࣜࢹ࣮ࢩࣙ
ࣥヨ㦂ࡀᐇ᪋ࡉࢀࡓ⤖ᯝ㸪᪋タෆ࠾ࡼࡧ᪋タ㛫ᕪࡀᑡ࡞ࡃ㸪ỗ⏝ᛶ࡟ඃࢀ࡚࠸
ࡿヨ㦂ἲ࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓ㸬ࡲࡓ㸪㞴⁐ᛶࡢࡓࡵホ౯࡛ࡁ࡞࠿ࡗࡓ⿕㦂≀㉁ࢆ㝖ࡃ࡜㸪ROS ࢔ࢵࢭ࢖࡟ࡼࡿගẘᛶ≀㉁ࡢ㝧ᛶ᳨ฟ⋡ࡣ 100%࡛࠶ࡾ㸪ഇ㝜ᛶ⤖ᯝࡀ࡞࠸ࡇ࡜࠿ࡽ㸪ROS ࢔ࢵࢭ࢖ࡣගẘᛶ࣏ࢸࣥࢩࣕࣝࡢホ౯࡟᭷⏝࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓ㸬ࡇࢀࡽࡢࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡟ᑐࡍࡿ➨୕⪅ホ౯࡟࠾࠸࡚
ࡶྠᵝࡢホ౯ࡀ࡞ࡉࢀࡓ㸬ࡇࡢࡼ࠺࡞⫼ᬒࡢࡶ࡜㸪JaCVAM ࡣ OECD ࡀᐃࡵࡓᅜ㝿ⓗ࡞ᇶ‽㸦2005 ᖺ㸧࡟ᚑࡗ࡚㸪ROS ࢔ࢵࢭ࢖ࡢࣂࣜࢹ࣮ࢩࣙࣥ≧ἣ࡟㛵ࡋ࡚⛉Ꮫⓗ࡞ホ౯ࡸ᳨ドࢆ⾜࠺ࡓࡵࡢᑓ㛛ᐙ࡟ࡼࡿ⊂❧ࡋࡓጤဨ఍ࢆタࡅࡓ㸬ጤဨ఍ࡣ 2013 ᖺ 2 ᭶࡟➨ 1ᅇࡢ఍ྜࢆᣢࡕ㸪ࡑࡢᚋ 5 ḟ࡟ரࡗ࡚ホ౯ሗ࿌᭩ࡢぢ┤ࡋࢆ㔜ࡡ࡚ࡁࡓ㸬ᮏホ౯ሗ࿌᭩ࡣጤဨ఍࡟ࡼࡿホ౯࡜⤖ㄽࢆせ⣙ࡋࡓࡶࡢ࡛࠶ࡿ㸬ᙜጤဨ఍ࡣ⤖ㄽ࡜ࡋ࡚㸪ROS ࢔ࢵࢭ࢖ࡢ෌⌧ᛶ࡜ண ᛶࡣ໬Ꮫ≀㉁ࡢ⥲ྜⓗ࡞ගẘᛶホ౯ୖ㸪༑ศ࡟㐺ᛂ࡛ࡁࡿ࢔ࢵࢭ࢖᪉ἲ࡛࠶ࡿ࡜⤖ㄽࡋࡓ㸬௒ᚋ㸪ROS࢔ࢵࢭ࢖࡟࠾࠸࡚㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜ㸪ḟࡢẁ㝵࡜ࡋ࡚ 3T3 ගẘᛶヨ㦂㸦OECD ヨ㦂࢞࢖ࢻࣛ࢖ࣥ#432㸧➼ࡢ in vitro ヨ㦂ࡢ㐺⏝ࡀ᥎ዡࡉࢀࡿࡀ㸪㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣ᭦࡞ࡿື≀ヨ㦂ࡸࡑࡢ௚ࡢヨ㦂ࡣ୙せ࡛࠶ࡿ࡜ᖐ⤖࡛ࡁࡿࡇ࡜࡟ࡼࡾ㸪ගẘᛶホ౯࡟ᚲせ࡜࡞ࡿ᫬㛫ࡸ㈝⏝ࢆ㍍ῶࡋ㸪ᐇ㦂ື≀ࡢ౑⏝ࢆ๐ῶࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜⪃࠼ࡽࢀࡿ㸬ࡲࡓ㸪ᮏ࢔ࢵࢭ࢖ࡢண ᛶ࣭෌⌧ᛶࡣ㸪་⸆ရࡢගẘᛶヨ㦂ἲࡢ୍⎔࡜ࡋ࡚㸪ࡑࡢ๰⸆ึᮇẁ㝵࠾ࡼࡧ㛤ⓎࡢពᛮỴᐃ࡟⏝࠸ࡿ஦ࢆᨭᣢ࡛ࡁࡿࡶࡢ࡛࠶ࡿ㸬ࡉࡽ࡟㸪᭱㏆ࡢ᳨ウ࡟࠾࠸࡚㸪ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿΰྜ≀࠾ࡼࡧศᏊ㔞୙᫂
ヨᩱ࡬ࡢ㐺⏝ྍ⬟ᛶࡀ♧ࡉࢀ㸪໬⢝ရཎᩱ㸪୍⯡໬Ꮫ≀㉁࡞ࡽࡧ࡟㎰⸆ࡢගᏳ඲ᛶ࡟㛵ࡍࡿࣁࢨ࣮ࢻ᳨ฟἲ࡜ࡋ࡚ ROS ࢔ࢵࢭ࢖࡬ࡢᮇᚅࡀ㧗ࡲࡗ࡚࠸ࡿ㸬511

 ヨ㦂᪉ἲࡢ⛉Ꮫⓗ⫼ᬒ࣭᰿ᣐ࠾ࡼࡧつไୖࡢ఩⨨࡙ࡅ
1.1

⛉Ꮫⓗ⫼ᬒ

 ගᏳ඲ᛶ࡜ࡣ㸪ගࢆ௓ࡋ࡚Ⓨ⑕ࡍࡿ໬Ꮫ≀㉁ㄏⓎᛶࡢẘᛶ཯ᛂ࡛࠶ࡾ㸪ᅗ 1(A)࡟♧ࡋࡓࡼ࠺࡟ග࡟ࡼࡗ࡚໬Ꮫ≀㉁ࡀບ㉳ࡍࡿࡇ࡜ࡀࢺ࣮ࣜ࢞࡜࡞ࡗ࡚࠸ࡿ㸬ࡇࡢࡼ࠺࡞ẘᛶࢆ♧ࡍ໬ྜ≀ࢆᅗ 1(B)࡛ࡣບ㉳ගẘᛶ໬ྜ≀࡜ᐃ⩏ࡋ࡚࠸ࡿࡀ㸪ࡇࡢࡼ࠺࡞≀㉁ࡣ㸪ග่⃭ᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶ㸪ග㑇ఏẘᛶ㸪ගࡀࢇཎᛶࢆㄏᑟࡍࡿྍ⬟ᛶࡀ࠶ࡿ㸬໬Ꮫ≀㉁ࡢගᏳ඲ᛶࢆホ౯ࡍࡿࡓࡵ࡟ᵝࠎ࡞࢔ࢵࢭ࢖ἲ㸦in vitro ࠾ࡼࡧ in vivo㸧ࡀ㛤Ⓨࡉࢀ࡚࠾ࡾ㸪ࡇࢀࡽ࡟ࡣ㸪(i) in silico ࢫࢡ࣮ࣜࢽࣥࢢࢶ࣮ࣝ㸪(ii) ග໬Ꮫⓗ≉ᛶࢆ୰ᚰ࡜ࡋࡓศᏊ≀ᛶホ౯㸪(iii) ྛ✀ẘᛶ཯ᛂ࡟≉␗ⓗ࡞ග⏕≀໬Ꮫⓗ࢔ࢵࢭ࢖᪉ἲ࡞࡝ࡀྵࡲࢀ࡚࠸ࡿ (Onoue et al., 2009)㸬
in silico ホ౯ࢩࢫࢸ࣒࡜ࡋ࡚ࡣ㸪౛࠼ࡤ DEREK ࡸ HOMO-LUMO Gap ➼ࡀ▱ࡽࢀ࡚࠸ࡿ㸬ග໬Ꮫⓗ≉ᛶホ౯ࢶ࣮ࣝࡣᐇ㝿࡟ྜᡂࡉࢀࡓ་⸆ရೃ⿵໬ྜ≀ࡢศᏊ≀ᛶࢆᣦᶆ࡜ࡋࡓග
ឤཷᛶศᯒࢆ୺య࡜ࡋ㸪⸆๣ᛶග⥺㐣ᩄ⑕ࡢㄏⓎ࡟ᐤ୚ࡋᚓࡿග໬Ꮫⓗ཯ᛂࡢ᭷↓࡟ࡘ࠸࡚㧗࠸ࢫ࣮ࣝࣉࢵࢺ࡛♧၀ࡍࡿࡇ࡜ࡀฟ᮶ࡿ㸬ගẘᛶࡢᶵᗎࢆ⪃࠼ࡿ࡜ࡁ㸪᭱ࡶ㔜せ࡞ࢺ࣮ࣜ࢞࡜࡞ࡿࡢࡣኴ㝧ගࡢ྾཰㸪ࡑࡋ࡚ࡑࢀ࡟క࠺໬ྜ≀ࡢບ㉳࡛࠶ࢁ࠺㸬ࡇࡢほⅬ࠿ࡽ㸪ࠕኴ㝧ගࡢ྾཰ࡋࡸࡍࡉࠖࡢᣦᶆ࡛࠶ࡿ UV/VIS ྾཰≉ᛶࡀ་⸆ရ㛤Ⓨ࡟࠾ࡅࡿගᏳ඲ᛶホ౯࡟࠾࠸࡚᭷⏝࡛࠶ࡿࡇ࡜ࡀ Henry ࡽ࡟ࡼࡗ࡚ᥦ᱌ࡉࢀࡓ㸬Henry ࡽࡢሗ࿌࡟ࡼࢀࡤ㸪ࣔࣝ྾ගಀᩘ 1,000 M-1㺃cm-1 ௨ୗࡢ໬ྜ≀ࡣගẘᛶࣜࢫࢡࡀప࠸ (Henry et al., 2009)㸬ࡉࡽ࡟㸪Bauer ࡽࡣࡇࡢ㜈್࡟ࡘ࠸࡚ࡼࡾከࡃࡢ໬Ꮫ≀㉁ࢆᑐ㇟࡟⢭ᰝࡋ㸪ࡑࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ドࡋ࡚࠸ࡿ (Bauer et al., 2014)㸬୍᪉㸪UV/VIS ྾཰≉ᛶࡔࡅ࡛࡞ࡃ㸪ບ㉳࢚ࢿࣝࢠ࣮࡟
ࡼࡿග໬Ꮫⓗ཯ᛂᛶࢆ┤᥋ホ౯ࡍࡿࡇ࡜ࡀྍ⬟࡜࡞ࢀࡤ㸪ࡼࡾᐇ㉁ⓗ࡞ගẘᛶண࡟ᐤ୚࡛ࡁࡿྍ⬟ᛶࡀ࠶ࡿ㸬ࡑࡇ࡛㸪Onoue ࡽࡣࡇࡢほⅬ࠿ࡽග↷ᑕୗ࡟࠾ࡅࡿ⸆≀࠿ࡽࡢ ROS⏘⏕ࢆᣦᶆ࡜ࡋࡓ᪂ࡓ࡞ගẘᛶணࢶ࣮࡛ࣝ࠶ࡿ ROS ࢔ࢵࢭ࢖ࢆ 2006 ᖺ࡟⪃᱌ࡋࡓ(Onoue and Tsuda, 2006)㸬ࡍ࡛࡟ୖᕷࡉࢀ࡚࠸ࡿ」ᩘࡢ་⸆ရࢆࡑࢀࡒࢀ ROS ࢔ࢵࢭ࢖࡛ホ౯ࡋࡓ࡜ࡇࢁ㸪๪స⏝ሗ࿌࡜ࡋ࡚ග⥺㐣ᩄ⑕ࡀሗ࿌ࡉࢀ࡚࠸ࡿ໬ྜ≀⩌ࡣ ROS ࢆ⏘⏕ࡍࡿഴྥ࡟࠶ࡾ㸪୍᪉㸪ᵝࠎ࡞ࢫࢡ࣮ࣜࢽࣥࢢ࡛ගᏳ඲ᛶࡢ㧗࠸࡜⪃࠼ࡽࢀࡿ໬ྜ≀⩌࠿ࡽࡢ ROS ⏘⏕ࡣᴟࡵ࡚㝈ᐃࡉࢀࡓࡶࡢ࡛࠶ࡗࡓ㸬ࡍ࡞ࢃࡕ㸪ROS ࢔ࢵࢭ࢖ࡣගẘᛶࣜࢫࢡࢆணࡍࡿ࠺࠼࡛᭷⏝࡞ࢶ࣮ࣝ࡜࡞ࡿྍ⬟ᛶࡀ♧၀ࡉࢀ㸪ᮏ࢔ࢵࢭ࢖ࡢࣁ࢖ࢫ࣮ࣝࣉࢵࢺ

ࢫࢡ࣮ࣜࢽࣥࢢ⣔࡬ࡢᛂ⏝᳨ウࡢ⤖ᯝ㸪ࢫ࣮ࣝࣉࢵࢺࡢᨵၿ࡜࢔ࢵࢭ࢖ࡢᑠࢫࢣ࣮ࣝ໬࡟
ᡂຌࡋ࡚࠸ࡿ (Onoue et al., 2008a)㸬ROS ࢔ࢵࢭ࢖ࡢ฼Ⅼ࡜ࡋ࡚ࡣ㸪໬Ꮫ≀㉁ࡢග໬Ꮫⓗ཯ᛂᛶࢆ㎿㏿࠿ࡘ⡆౽࡟ᐃ࡛ࡁࡿࡇ࡜ࡀᣲࡆࡽࢀࡿࡀ㸪ࡑࡢ཯㠃㸪ᮏ࢔ࢵࢭ࢖ࢹ࣮ࢱࡣග

Ᏻ඲ᛶࡔࡅ࡛ࡣ࡞ࡃ㸪ගᏳᐃᛶ࡞࡝࡟ၥ㢟ࡀ࠶ࡿ໬ྜ≀ࡶྵࡵ࡚㝧ᛶ࡜ุูࡋ࡚ࡋࡲ࠺ࡇ
࡜ࡀㄢ㢟ࡢ୍ࡘ࡛࠶ࡾ㸪ࡇࢀࡣග໬Ꮫⓗ཯ᛂᛶホ౯ἲ࡛࠶ࡿࡀᨾࡢㄢ㢟࡛࠶ࡿ㸬612

 ᮏᩥ᭩࡟࠾࠸࡚㸪ගᏳ඲ᛶ࡜ࡣ໬Ꮫ≀㉁࡟᥋ゐ࠶ࡿ࠸ࡣᦤྲྀࡋࡓᚋ࡛㸪ග࡬ࡢ᭚㟢࡟㛵㐃ࡋ࡚Ⓨ⑕ࡍࡿ᭷ᐖస⏝඲⯡ࢆᑐ㇟࡜ࡋ㸪ᚑ᮶ࡼࡾ㸪ගẘᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶ㸪ග㑇ఏẘᛶ㸪ගࡀࢇཎᛶࡢ 4 ✀ࡢ␗࡞ࡿస⏝ࡀྵࡲࢀࡿ㸬ගᏳ඲ᛶࡣ㸪ᑐ㇟࡜࡞ࡿ໬Ꮫ≀㉁ࡢග໬
Ꮫⓗ≉ᛶ㸪㠀⮫ᗋヨ㦂ࡢࢹ࣮ࢱ࠾ࡼࡧ⮫ᗋᏳ඲ᛶ᝟ሗࢆࡩࡲ࠼࡚⤫ྜⓗ࡟ホ౯ࡉࢀࡿ㸬ගẘᛶࡣග่⃭ᛶ࡜ྠ⩏࡛㸪ග↷ᑕ࡟ࡼࡗ࡚⏘⏕ࡉࢀࡿග཯ᛂᛶ≀㉁࡟ᑐࡍࡿᛴᛶࡢ⤌⧊཯ᛂ࡛࠶ࡿ㸬ග⥺㐣ᩄ⑕࡜ࡣ㸪ග↷ᑕ࡟ࡼࡾច㉳ࡉࢀࡿ⤌⧊཯ᛂ࡟ᑐࡋ㸪᫬ᢡ౑⏝ࡉࢀࡿ୍⯡⏝ㄒ࡛࠶ࡾ㸪ᗈ⩏ࡢගẘᛶ࡜ྠࡌព࿡࡛⏝࠸ࡽࢀࡿࡇ࡜ࡶ࠶ࡿࡀ㸪ගẘᛶ࡜ග࢔ࣞࣝࢠ
࣮ᛶࢆ᫂☜࡟༊ูࡍࡿᚲせࡀ࠶ࡿሙྜ࡟౑⏝ࡍࡿࡇ࡜ࡣ㐺ษ࡛ࡣ࡞࠸㸬
ROS ࢔ࢵࢭ࢖ࣂࣜࢹ࣮ࢩ࣐ࣙࣥࢿࢪ࣓ࣥࢺࢳ࣮࣒ࡣ JaCVAM ᣦᑟୗ࡛」ᩘࡢཧຍ᪋タ࡛ 2 ✀㢮ࡢᨃఝኴ㝧ග↷ᑕ⿦⨨ࢆ⏝࠸ࡓࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 2 ヨ㦂ࢆᐇ᪋ࡋ㸪ROS ࢔ࢵࢭ࢖ࡢᢏ⾡⛣㌿ᛶ㸦transferability㸧㸪ሀ∼ᛶ㸦robustness㸧࡞ࡽࡧ࡟ண

ᛶ㸦prediction capacity㸧ࢆ᳨ドࡋࡓ㸬ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂⤖ᯝࢆㄽᩥ࡜ࡋ࡚ᢞ✏ࡋ(Onoue et al., 2013a; Onoue et al.,2014a)㸪ヲ⣽࡞ࢹ࣮ࢱࢆྵࡴࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡞ࡽࡧ࡟ヨ㦂ἲࣉࣟࢺࢥ࣮ࣝࢆ JaCVAM
࣮࣒࣮࣍࣌ࢪୖ࡟බ㛤ࡋࡓ㸦ROS assay validation management team, 2013a, 2013b, 2013c㸧㸬
ROS ࢔ࢵࢭ࢖ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࢆホ౯ࡋࡓ➨୕⪅ホ౯ጤဨ఍ࡢሗ࿌᭩ࡶ JaCVAM ࣮࣒࣮࣍࣌ࢪୖ࡟බ㛤ࡉࢀ㸦Spielmann et al., 2013㸧㸪ࡑࡢせ⣙ࢆ Appendix 1 ࡟♧ࡋࡓ(Appendix
1: Executive Summary of ͆Peer Review Panel Evaluation of the Reactive Oxygen Species (ROS)
Photosafety Assay”).

1.2

⛉Ꮫⓗ᰿ᣐ

 ࡝ࡢࡼ࠺࡞Ⓨ⑕ᶵᗎࡢගẘᛶ཯ᛂ࡛ࡶ㸪ࡑࡢⓎ⑕ᶵᗎࡢୖὶ࡟ࡣග໬Ꮫ཯ᛂࡀᏑᅾࡍࡿ㸬
1817 ᖺ࡟ᥦၐࡉࢀࡓග໬Ꮫ➨୍ἲ๎ (ࡲࡓࡣ Grotthuss-Draper law) ࡛ࡣ ͆ධᑕࡋࡓගࡢ࠺
ࡕ㸪྾཰ࡉࢀࡓࡶࡢࡔࡅࡀ཯ᛂ࡟㛵ࢃࡿ͇ ࡜࠶ࡾ㸪ࡍ࡞ࢃࡕග྾཰ࡍࡿ໬ྜ≀ࡢࡳࡀගẘᛶ཯ᛂࢆ♧ࡋᚓࡿࡇ࡜ࡀ㏙࡭ࡽࢀ࡚࠸ࡿ㸬࢜ࢰࣥᒙࢆ㏻ࡗ࡚ᆅ⾲࡟฿㐩࡛ࡁࡿኴ㝧ගࡢἼ㛗ࡣ୺࡟ 290̽700 nm ⛬ᗘ࡛࠶ࡿࡀ㸪300 nm ࡼࡾࡶ▷Ἴ㛗࡛ẚ㍑ⓗ࢚ࢿࣝࢠ࣮ࡢ㧗࠸ගࡢ┿⓶฿㐩⋡ࡣࢃࡎ࠿ 10% ⛬ᗘ࡛࠶ࡿ㸬୍᪉㸪㛗Ἴ㛗ഃ࡟࡞ࡿ࡟ࡘࢀ࡚࢚ࢿࣝࢠ࣮ࡣᑠࡉࡃ
࡞ࡿࡶࡢࡢ㸪⓶⭵㏱㐣ᛶࡣ㧗ࡃ࡞ࡾ㸪ࡇࢀࡽࡀ⸆๣ᛶග⥺㐣ᩄ⑕ࡢࢺ࣮ࣜ࢞࡜࡞ࡿࡇ࡜ࡶ
ከ࠸㸬యෆ࡟ྲྀࡾ㎸ࡲࢀࡓගẘᛶ≀㉁ࡣࡲࡎ⓶⭵⤌⧊࡟฿㐩ࡋ㸪⸆๣ࡢศᏊෆ chromophore㸪࠶ࡿ࠸ࡣ௦ㅰ࡟ࡼࡗ࡚⋓ᚓࡉࢀࡓ chromophore ࡀ⓶⭵῝㒊ࡲ࡛฿㐩ࡋࡓග࡟ࡼࡗ࡚↷ᑕࡉࢀࡿ࡜㸪ᇶᗏ≧ែࡢ S0 ࠿ࡽບ㉳୍㔜㡯≧ែ S1 ࡟ບ㉳ࡉࢀࡿ㸦ᅗ 1(A)㸧㸬ບ㉳୍㔜㡯≧ែࡢᑑ࿨ࡣ▷ࡃ㸪ࡍ࡞ࢃࡕ⺯ගࢆⓎࡋ࡚┤ࡕ࡟ᇶᗏ≧ែ S0 ࡟ᡠࡿ࠿㸪㡯㛫஺ᕪ࡟ࡼࡾບ
㉳୕㔜㡯≧ែ T1 ࡟㑄⛣ࡍࡿ㸬ບ㉳୕㔜㡯≧ែ࡟࠶ࡿ໬ྜ≀ࡣࡾࢇගࢆⓎࡋ࡚ᇶᗏ≧ែ S0࡟ᡠࡿ㸬ࡇࡢࡼ࠺࡟ගࢆ྾཰ࡋ࡚ບ㉳ࡉࢀࡓศᏊࡣ㏿ࡸ࠿࡟ࡶ࡜ࡢᇶᗏ≧ែ࡟ᡠࡿ≉ᛶࡀ࠶ࡿࡀ㸪ࡇࡢ㐣⛬ࡀᴟࡵ࡚ࢫ࣒࣮ࢬ࡟㉳ࡇࢀࡤ྾཰ࡉࢀࡓග࢚ࢿࣝࢠ࣮࡟ࡼࡿගẘᛶ཯ᛂ࡬ࡢᐤ୚ࡣ㝈ᐃࡉࢀࡓࡶࡢ࡜࡞ࡿ㸬ࡋ࠿ࡋ㸪ᇶᗏ≧ែ࡛ࡣࡲࡗࡓࡃ໬Ꮫ཯ᛂࢆࡋ࡞࠸᮲௳

713࡛ࡶ㸪㧗࠸ග࢚ࢿࣝࢠ࣮ࢆ⋓ᚓࡋࡓບ㉳ศᏊࡣ㸪ࡑࡢ࢚ࢿࣝࢠ࣮ࢆ㥑ືຊ࡜ࡋ࡚⤖ྜࡢゎ⿣ࡸ⏕ᡂࡲࡓࡣ⤌ࡳ᥮࠼࡞࡝ࡢ໬Ꮫ཯ᛂࢆ㉳ࡇࡍࡇ࡜ࡀ࡛ࡁࡿ㸬ࡑࡢࡼ࠺࡞㐣⛬ࢆග໬Ꮫ㐣⛬࡜࠸࠸㸪ࣛࢪ࢝ࣝ཯ᛂ࡛࠶ࡿ Type I ཯ᛂ࡜㸪singlet oxygen ཯ᛂ࡛࠶ࡿ Type II ཯ᛂ࡜࡟ศࡅࡽࢀࡿ㸬ࡇࢀࡽࡣ⸆๣ᛶග⥺㐣ᩄ⑕ࡢⓎ⑕࡟࠾࠸࡚ึᮇ࡟ㄆࡵࡽࢀ㸪ࡍ࡞ࢃࡕ UV/VISࡢ᭚㟢࡟ࡼࡗ࡚ບ㉳ࡉࢀ㸪ࡑࡢ㝿࡟ᚓࡽࢀࡓບ㉳࢚ࢿࣝࢠ࣮ࡣ⏕యෆ≀㉁ࡸ㓟⣲ศᏊ࡟㌿⛣ࡋࡓࡾ㸪࠶ࡿ࠸ࡣ໬ྜ≀ࡑࡢࡶࡢࡀ⏕యෆ≀㉁࡜⤖ྜࡋ࡚ග௜ຍ≀ࢆᙧᡂࡍࡿ㸬ࡲࡓ㸪㓟⣲ศᏊࡣບ㉳࢚ࢿࣝࢠ࣮ࡢ࢔ࢡࢭࣉࢱ࣮࡜ࡋ࡚ᶵ⬟ࡋ㸪ࡑࢀ࡟క࠸⏘⏕ࡉࢀࡓ singlet
oxygen ࡸ superoxide ➼ࡢάᛶ㓟⣲✀࡟ࡼࡿ⏕యෆ≀㉁ࡢ㓟໬཯ᛂࡀ⸆๣ᛶග⥺㐣ᩄ⑕ࡢ
Ⓨ⑕ཎᅉࡢ୍ࡘ࡜ࡋ࡚⪃࠼ࡽࢀ࡚࠸ࡿ㸦ᅗ 1(B㸧㸧㸬ࡇࢀࡽࡢග໬Ꮫ཯ᛂࡢᶆⓗࡀ⣽⬊⭷ୖࡢྛ✀⏕యᡂศ࡛࠶ࡿሙྜ࡟ࡣග่⃭ᛶࢆㄏⓎࡋ㸪ࡲࡓ DNA ࡢ㓟໬࠶ࡿ࠸ࡣሷᇶಟ㣭࡟ࡼࡗ࡚ග㑇ఏẘᛶࡸගࡀࢇཎᛶࡀⓎ⌧ࡍࡿ㸬ບ㉳ࡉࢀࡓ⸆≀ࡀࣁࣉࢸࣥ࡜࡞ࡾࢱࣥࣃࢡ㉁࡜ග௜ຍ≀ࢆᙧᡂࡋࡓ㝿࡟ࡣ㸪ච␿ཎᛶࢆ♧ࡍࡇ࡜࡟࡞ࡾ㸪᭱⤊ⓗ࡟ග࢔ࣞࣝࢠ࣮ᛶ཯ᛂ
ࢆច㉳ࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡿ㸬ࡇࡢࡼ࠺࡟㸪⸆≀ࡀບ㉳ࡉࢀࡓᚋ࡟཯ᛂࡍࡿศᏊ✀࡟ࡼࡗ
࡚Ⓨ⌧ࡍࡿẘᛶ཯ᛂࡀࡑࢀࡒࢀ␗࡞ࡿ㸬ࡇࢀࡽࡢᶵᗎ࡟ຍ࠼࡚㸪ບ㉳࢚ࢿࣝࢠ࣮ࡸάᛶ㓟⣲✀࡟ࡼࡗ࡚⸆≀ࡑࡢࡶࡢࡀศゎࡉࢀ㸪ẘᛶ≀㉁ࢆ⏕ᡂࡍࡿࢣ࣮ࢫࡶ᝿ᐃࡉࢀ㸪ẘᛶᶵᗎࡣከᵝ࠿ࡘ」㞧࡛࠶ࡿ㸬ࡇࡢࡼ࠺࡞ග໬Ꮫⓗ཯ᛂࡀගẘᛶ཯ᛂࡢࢺ࣮ࣜ࢞࡟࡞ࡗ࡚࠸ࡿࡇ
࡜ࢆ⪃៖ࡍࢀࡤ㸪ROS ࢔ࢵࢭ࢖ࢆගẘᛶホ౯ࢶ࣮ࣝࡢ୍ࡘ࡜ࡋ࡚฼⏝ࡍࡿࡇ࡜ࡣጇᙜ࡜⪃
࠼ࡽࢀࡿ㸬

ᅗ 1

ගẘᛶᶵᗎ㸦௬ㄝ㸧㸬(A) ග࡟ࡼࡿ໬Ꮫ≀㉁ࡢບ㉳࡜ᇶᗏ≧ែ࡬ࡢ㑄⛣㸬(B) ບ㉳໬ྜ≀ࡢᶆⓗ࡜࡞ࡿ⏕యෆศᏊ࡜ගẘᛶ཯ᛂ㸬(ᑿୖ, 2011)

8
14

1.3

つไୖࡢ఩⨨࡙ࡅ

๓㏙ࡢࡼ࠺࡟㸪ග↷ᑕ࡟ࡼࡗ࡚ບ㉳ගẘᛶ≀㉁ࢆⓎ⏕ࡍࡿ໬ྜ≀ࡣ㸪ග่⃭ᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶ㸪ග㑇ఏẘᛶࡲࡓࡣගࡀࢇཎᛶࢆㄏᑟࡍࡿྍ⬟ᛶࡀ࠶ࡿࡶࡢࡢ㸪ICH ࡟࠾࠸࡚సᡂ
ࡉࢀࡓ་⸆ရࡢගᏳ඲ᛶホ౯࡟㛵ࡍࡿ࢞࢖ࢻࣛ࢖ࣥࡢ㐺⏝⠊ᅖࡣ㸪ග่⃭ᛶ࡜ග࢔ࣞࣝࢠ
࣮ᛶ࡟㝈ᐃࡋ࡚࠸ࡿ㸬ගᏳ඲ᛶホ౯࡟㛵ࡋ࡚ࡣࡇࢀࡲ࡛ 2002 ᖺ࡟Ḣᕞ་⸆ရᑂᰝᗇ
(EMEA; ⌧ EMA)/Ḣᕞ་⸆ရጤဨ఍ (CPMP)㸪⩣ᖺࡢ 2003 ᖺ࡟ࡣ⡿ᅜ㣗ရ་⸆ရᒁ
(FDA)/་⸆ရホ౯◊✲ࢭࣥࢱ࣮(CDER)㸪ࡉࡽ࡟ 2004 ᖺ࡟ࡣ⤒῭༠ຊ㛤Ⓨᶵᵓ (OECD)࠿
ࡽホ౯᪉ἲࡀᥦ♧ࡉࢀ࡚࠸ࡿࡀ㸪ࡇࢀࡽ 3 ࡘࡢ࢞࢖ࢻࣛ࢖ࣥ/࢞࢖ࢲࣥࢫࡢඹ㏻Ⅼ࡜ࡋ࡚㸪໬ྜ≀ࡢග໬Ꮫⓗ≉ᛶࢆホ౯ࡍࡿᚲせᛶࡀグ㏙ࡉࢀ࡚࠸ࡿࡇ࡜ࡀᣲࡆࡽࢀࡿ㸬ࡇࢀࡽࡢ࢞
࢖ࢻࣛ࢖ࣥ/࢞࢖ࢲࣥࢫ࡛ࡣึᮇࢫࢡ࣮ࣜࢽࣥࢢ࡜ࡋ࡚ UV/VIS ྾཰≉ᛶࡀᥦ᱌ࡉࢀ࡚࠸
ࡿ㸬OECD ࢞࢖ࢻࣛ࢖࡛ࣥࡣホ౯⣔࡟࠾ࡅࡿࣔࣝ྾ගಀᩘ࡟㛵ࡍࡿ㜈್ࢆ 10 M-1㺃cm-1 ࡜タᐃࡋ࡚࠾ࡾ㸪ᐇ㉁ⓗ࡟ࡣ඲࡚ࡢ໬ྜ≀ࡀࡇࡢ㜈್ࢆ㉸࠼ࡿ㸬ࡑࢀᨾ㸪ࡼࡾ⌧ᐇⓗ࡞㜈್ࡢタᐃࢆ┠ⓗ࡜ࡋࡓ◊✲ࡀ⾜ࢃࢀ㸪Henry ࡽࡣ᪤▱ගẘᛶ་⸆ရࢆᑐ㇟࡜ࡋ࡚ග྾཰≉ᛶࢆ⢭ᰝࡋࡓ⤖ᯝ㸪⤒㦂๎ⓗ࡟ࣔࣝ྾ගಀᩘ 1,000 M-1㺃cm-1 ࡀࡼࡾ㐺ษ࡞㜈್࡛࠶ࡾ㸪ࡇࢀࢆ㉸࠼࡞࠸་⸆ရ࡟ࡘ࠸࡚ࡣගᏳ඲ᛶホ౯ࢆᐇ᪋ࡍࡿᚲせᛶࡣప࠸ࡇ࡜ࢆ♧ࡋ࡚࠸ࡿ(Henry et al., 2009)㸬EMA ࡣ 2010 ᖺ࡟⮬ࡽࡢ࢞࢖ࢲࣥࢫ࡟㛵ࡍࡿ Question and Answers ࢆබ⾲ࡋ㸪ࡇࡢ⪃࠼ࢆ㏣㝶ࡋ࡚࠸ࡿ㸬 ICH ࡟࠾࠸࡚་⸆ရࡢගᏳ඲ᛶホ౯ࡀᏳ඲ᛶ࡟㛵ࡍࡿࢺࣆࢵࢡ࡜ࡋ࡚ 2010 ᖺ࡟᥇ᢥࡉࢀ㸪EWG ࡀⓎ㊊ࡋ㸪3 ᖺ㛫ࡢ᳨ウᮇ㛫ࢆ⤒࡚㸪2013 ᖺ 11 ᭶࡟ගᏳ඲ᛶホ౯࡟㛵ࡍࡿ࢞࢖ࢻࣛ࢖ࣥࡢ Step 4 ᩥ᭩ࡀྜពࡉࢀࡓ㸬ICH ࢞࢖ࢻࣛ࢖ࣥ࡟࠾
࠸࡚ගẘᛶホ౯ࡢせྰࢆุ᩿ࡍࡿึᮇホ౯࡟࠾࠸࡚㸪ࣔࣝ྾ගಀᩘࡀ 1000 M-1㺃cm-1 ࢆ㉸࠼ࡿ≀㉁࡛࠶ࡗ࡚ࡶ໬Ꮫⓗග཯ᛂᛶホ౯⣔࡟࠾࠸࡚㝜ᛶ࡛࠶ࢀࡤ㸪ࡉࡽ࡞ࡿගẘᛶホ౯ࡸ⮫ᗋヨ㦂࡟࠾ࡅࡿග㜵ᚚᥐ⨨ࡣ୙せ࡜ࡉࢀ࡚࠸ࡿ㸬ROS ࢔ࢵࢭ࢖ࡣ໬Ꮫⓗග཯ᛂᛶ࠿ࡽ⿕㦂≀㉁ࡢගẘᛶࡀ㝜ᛶ࡛࠶ࡿࡇ࡜ࢆ☜ㄆ࡛ࡁࡿ᪉ἲ࡜ࡋ࡚㸪་⸆ရࡢගᏳ඲ᛶホ౯࡟฼⏝ࡉ
ࢀ࡚࠸ࡿ㸬

9
15

⾲ 1

Ḣ⡿ࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ
EMEA/CPMP࢞࢖ࢻࣛ࢖ࣥ

Guidance on
photosafety
testing

FDA/CDER
Guidance forindustry
photosafety
testingOECD

ICH

In vitro 3T3 NRU
phototoxicity test

Photosafety
evaluation ofpharmaceuticals(S10)ගẘᛶホ౯ࡀᚲせ࡜࡞ࡿ᮲௳࣭໬Ꮫ≀㉁඲⯡ ࣭඲࡚ࡢཎ⸆࠾ࡼ ࣭UV/VIS ྾཰ࢆ ࣭᪂つ་⸆ရ᭷࣭ࣂ࢖࢜ࢸࢡࣀࣟ ࡧ〇๣ᡂศ♧ࡍ໬Ꮫ≀㉁ຠᡂศ(API)ࢪ࣮་⸆ရ࣭እ⏝๣ࡣ〇๣࡛ ඲⯡࣭᪂つῧຍ๣
ࡢᐇ᪋ࢆ᥎ዡ
࣭⤒⓶ᢞ୚⏝⮫
ᑐ㇟໬ྜ≀ࡢ⠊ᅖᗋ〇๣㸦⓶⭵㈞
௜๣࡞࡝㸧࣭ග⥺ຊᏛ⒪ἲ
⏝〇๣
࣭290–700 nm ࣔ ࣭290–700 nm ࣔ ࣭290–700 nm ࣔ ࣭290–700 nm ࣔࣝ྾ගಀᩘ࡟
ࣝ྾ගಀᩘ࡟
ࣝ྾ගಀᩘ㸸10 ࣝ྾ගಀᩘ㸸1000 M-1㺃cm-1
㛵ࡍࡿグ㍕࡞
㛵ࡍࡿグ㍕࡞
M-1㺃cm-1 ௨ୖ௨ୖࡋࡋ͌UV/VIS ࡢ྾཰
UV/VIS ྾཰≉ᛶ࡟ࡼࡾ㸪཯ᛂᛶ
࡟ᐩࢇࡔศᏊ
✀ࢆᙧᡂࡍࡿ㸦ROS ࢔ࢵࢭ࢖㸧࣭ᒁᡤ㐺⏝࣭⓶⭵࣭║࡟㐺⏝࣭⓶⭵㺃║࡟༑ศ
ᢞ୚⤒㊰࡜యෆศ ࣭⓶⭵㺃║࡟฿㐩ࡶࡋࡃࡣศᕸ
㔞ศᕸ㐺⏝እᕸ࣭⓶⭵㺃║࡟ᙳ㡪ࢆ୚࠼ࡿࡶࡢ

ホ౯᪉ἲ

Photoirritation

Photoallergy

Photogenotoxicity

࣭3T3 NRU
࣭3T3 NRU
࣭3T3 NRU
࣭3T3 NRU
phototoxicity test phototoxicity test phototoxicity test phototoxicity test࣭࣐࢘ࢫ㸪ࣔࣝࣔ
ࢵࢺ㸪ࣈࢱ㸪࢘ࢧࢠࢆ⏝࠸ࡓ
in vivo ヨ㦂࣭ᨵⰋࡉࢀࡓ
࣭⤒⓶ᢞ୚་⸆ရ
LLNA ࢔ࢵࢭ
࡟࠾࠸࡚ࡶ㸪㠀
࢖ࡸ MEST ࢔⮫ᗋග࢔ࣞࣝ
ࢵࢭ࢖ࡀᑗ᮶㐺⏝እ㐺⏝እࢠ࣮ᛶヨ㦂ࡢ
ⓗ࡟᭷⏝࡟࡞
⤖ᯝࡢࣄࢺ࡬
ࡿྍ⬟ᛶࢆ♧
ࡢண ᛶࡣ୙
၀ࡍࡿࡢࡳ᫂࡜ࡋ࡚࠸ࡿ
࣭ග໬ᏛⓗᰁⰍయ ࣭ࢧࣝࣔࢿࣛ⳦㸪
␗ᖖㄏⓎᛶヨ㓝ẕ㸪V79 ⣽⬊ࢆ⏝࠸ࡓ in㦂࣭in vivo ࢥ࣓ࢵࢺ vitro ග㑇ఏẘ
㐺⏝እ
㐺⏝እ
ヨ㦂
ᛶヨ㦂
࣭ࢺࣛࣥࢫࢪ࢙ࢽ
ࢵࢡኚ␗ཎᛶ
ࣔࢹࣝ

1016

2 ヨ㦂ࡢࣉࣟࢺࢥࣝ࠾ࡼࡧุᐃᇶ‽
2.1

ᐃཎ⌮

 ஦๓ࡢ᳨ウ࡟࠾࠸࡚㸪ග↷ᑕࡋࡓ⸆≀࠿ࡽ⏘⏕ࡉࢀࡿ singlet oxygen ࡜ superoxide ࡢ 2✀ࡢ ROSᐃࡀ⢭ᗘࡢ㧗࠸ගᏳ඲ᛶホ౯࡟ᐤ୚ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓ (Onoue et al.,

2006&2008)㸬ࡑࡇ࡛㸪ࡇࡢ 2 ✀ࡢ ROS ⏘⏕ࢆホ౯ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋ࡚ ROS ࢔ࢵࢭ࢖ࡀᵓ⠏ࡉࢀࡓ㸬Singlet oxygen ࡣ p-nitrosodimethyl aniline ࡢኚⰍࢆᣦᶆ࡜ࡋ࡚ࡑࡢ⏘⏕ࢆホ౯࡛ࡁࡿ㸬ࡍ࡞ࢃࡕ㸪ບ㉳໬ྜ≀࠿ࡽ⏘⏕ࡉࢀࡓ singlet oxygen ࡣ཯ᛂᾮ୰ࡢ imidazole ⎔࡜཯ᛂࡋ࡚ trans-annular peroxide intermediate ࢆᙧᡂࡍࡿࡀ㸪ࡇࢀࡀ p-nitrosodimethyl anilineࢆ㓟໬ࡍࡿࡇ࡜࡛ኚⰍࢆࡶࡓࡽࡋ㸪440 nm ࡢ྾ග್ࡀῶᑡࡍࡿ㸬ࡇࡢ྾ග್ࡢῶᑡࡀ
singlet oxygen ࡢ⏕ᡂࢆ♧၀ࡍࡿࡶࡢ࡛࠶ࡿ㸬
Photoirradiated chemical + dissolved oxygen Ѝoxidized chemical + singlet oxygen
singlet oxygen + imidazole ĺ [peroxide intermediate] ĺ oxidized imidazole[peroxide intermediate] + p-nitrosodimethyl aniline ĺ p-nitrosodimethyl aniline + products
 Superoxide ࡶྠࡌࡃศගᏛⓗᡭἲ࡟ࡼࡗ࡚

ᐃࡀྍ⬟࡛࠶ࡾ㸪ᇶ㉁࡜ࡋ࡚ nitroblue

tetrazolium (NBT) ࢆ ౑⏝ ࡍࡿ 㸬ບ㉳ ໬ྜ ≀࠿ࡽ ⏘⏕ ࡉࢀࡿ superoxide ࡣ one-electrontransfer reaction ࡟ࡼࡾ NBT ࢆ㑏ඖࡋ㸪⏕ᡂࡉࢀࡿ monoformazan (NBT+) ࡣ 560 nm ࡟
UV ྾཰ࢆ᭷ࡍࡿ㸬ࡇࡢ྾ග್ࡢୖ᪼ࡀ superoxide ࡢ⏕ᡂࢆ♧၀ࡍࡿ㸬
Photoirradiated chemical + dissolved oxygen Ѝoxidized chemical + superoxide
superoxide + NBT ĺ O2 + NBT+
 ࡇࢀࡽࡢ཯ᛂࡣ photodynamic ࡟ಁ㐍ࡉࢀࡿࡀ㸪ග↷ᑕᙉᗘ㸪ග↷ᑕ᫬㛫㸪ࢧࣥࣉࣝᗘ㸪ඹᏑࡍࡿ᭷ᶵ⁐፹ࢆࡣࡌࡵ࡜ࡍࡿ✀ࠎࡢ࢔ࢵࢭ࢖᮲௳࡟ࡼࡗ࡚ࡶ኱ࡁࡃኚືࡀㄏⓎࡉࢀࡿࡓࡵ㸪⣽ᚰࡢὀពࡀᚲせ࡛࠶ࡿ㸬

2.2ᮦᩱ࠾ࡼࡧヨ⸆

2.2.1

ROS ࢔ࢵࢭ࢖⏝ヨ⸆㢮࡞ࡽࡧ࡟ࡑࡢㄪ〇1117

 NaH2PO4࣭2H2O (CAS#13472-35-0), Na2HPO4࣭12H2O (CAS#10039-32-4), dimethyl sulfoxide(DMSO,

CAS#67-68-5),

p-nitrosodimethyl

aniline(RNO,

CAS#138-89-6),

imidazole(CAS#288-32-4), and nitroblue tetrazolium (NBT, CAS#298-83-9) ࡣᕷ㈍ࡢヨ⸆ࢆ౑⏝ࡍࡿ.

2.2.2 20 mM sodium phosphate buffer (NaPB, pH7.4)
 3.8 mmol (593 mg) ࡢ NaH2PO4࣭2H2O ࡜㸪16.2 mmol (5.8 g) ࡢ Na2HPO4࣭12H2O ࢆ㔞ࡾྲྀࡾ㸪900 mL ࡢ⢭〇Ỉ࡟⁐ゎࡋࡓ㸬HCl ࡟ࡼࡗ࡚ pH7.4 ࡟ㄪᩚࡋ㸪⢭〇Ỉ࡛ 1 L ࡟࣓ࢫ࢔ࢵࣉࡋ࡚ࡼࡃΰ࿴ࡍࡿ㸬

2.2.3 200 PM p-nitrosodimethyl aniline Ỉ⁐ᾮ 20 μmol (3 mg) ࡢ p-nitrosodimethyl aniline ࢆ 100 mL ࡢ NaPB ࡟⁐ゎࡍࡿ㸬ㄪ〇ᚋࡣ㐽ගࣂ࢖࢔ࣝ࡟ධࢀ㸪෭ⶶᗜ࡟࡚ಖ⟶ࡍࡿ㸬

2.2.4

200 PM imidazole Ỉ⁐ᾮ 200 μmol (13.6 mg) ࡢ imidazole ࢆ 10 mL ࡢ NaPB ࡟⁐ゎࡋ㸪ࡉࡽ࡟ NaPB ࡛ 100 ಸᕼ㔘ࡍࡿ㸬ㄪ〇ᚋࡣ㐽ගࣂ࢖࢔ࣝ࡟ධࢀ㸪෭ⶶᗜ࡟࡚ಖ⟶ࡍࡿ㸬

2.2.5 400 PM NBT Ỉ⁐ᾮ 40 μmol (32.7 mg) ࡢ NBT ࢆ 100 mL ࡢ NaPB ࡛⁐ゎࡋࡓ㸬NBT ࡣ⁐ゎᗘࡀప࠸ࡓࡵ㸪㉸㡢Ἴฎ⌮ࡸ࣎ࣝࢸࢵࢡࢫ➼ࡢ⁐ゎ⿵ຓ᧯సࡀᚲせ࡜࡞ࡿࡇ࡜ࡀ࠶ࡿ㸬ㄪ〇ᚋࡣ㐽ගࣂ࢖࢔ࣝ࡟ධࢀ㸪෭ⶶᗜ࡟࡚ಖ⟶ࡍࡿ㸬

2.3

⿕㦂≀㉁

 ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡍࡿ㝿࡟ࡣ㸪 DMSO ࡛ 10 mM ࡢ stock solution ࢆ࢔ࢵࢭ࢖┤๓࡟ㄪ〇ࡋ㸪ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿ⤊⃰ᗘࡣ 200 μM ࡜ࡍࡿ㸬ගᏳᐃᛶ࡟ὀពࢆࡋࡘࡘ㸪࣎ࣝࢸࢵࢡࢫ࣑࢟ࢧ࣮࡟࡚ 5–10 ศ⛬ᗘΰ࿴ࡋ⁐ゎᛶࢆ☜ㄆࡍࡿࡇ࡜ࢆ᥎ዡࡍࡿࡀ㸪⿕㦂≀㉁ࡢ⁐ゎᗘࡀప࠸ሙྜ࡟ࡣ NaPB ࡛⁐ゎࡋ࡚ࡶࡼ࠸㸬ࡑࢀ࡛ࡶ࡞࠾⁐ゎࡋ࡞࠸ሙྜ࡟ࡣ㸪DMSO stock solution ࢆ DMSO ࡛ᕼ㔘ࡋ⁐ゎᛶࢆ☜ㄆࡋ࡚ ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡍࡿࡇ࡜
࡜ࡍࡿ㸬ᕼ㔘ࡍࡿሙྜ࡟ࡣ ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿ⤊⃰ᗘࡀ㸪20㸪50㸪࠶ࡿ࠸ࡣ 100 μM ࡜࡞ࡿࡇ࡜ࡀᮃࡲࡋ࠸ࡀࡇࡢ㝈ࡾ࡛ࡣ࡞࠸㸬ᕼ㔘ࡋࡓሙྜ࡟ࡣุᐃᇶ‽ࡀ㠀ᕼ㔘᫬࡜ኚࢃࡿ
ࡢ࡛ὀពࡀᚲせ࡛࠶ࡿ㸬࡞࠾㸪200 μM ࡢ⤖ᯝࡢุᐃࢆ⏝࠸ࡿࡇ࡜ࡀ᥎ዡࡉࢀ㸪⁐ࡅ࡞࠸ሙྜ࡟ࡣప⃰ᗘࢆ౑⏝ࡍࡿࡀ㸪⤖ᯝࡢุ᩿࡟ࡣὀពࡀᚲせ࡛࠶ࡿ㸬

12
18

2.4

㝧ᛶ࡞ࡽࡧ࡟㝜ᛶᑐ↷≀㉁ ROS ࢔ࢵࢭ࢖࡟࠾࠸࡚ࡣ㝧ᛶᑐ↷≀㉁࡜ࡋ࡚ quinine HCl (CAS#6119-47-7)㸪ࡑࡋ࡚㝜ᛶᑐ↷≀㉁࡜ࡋ࡚ sulisobenzone (CAS#4065-45-6) ࢆ⏝࠸ࡿࡇ࡜ࡀᮃࡲࡋ࠸㸬࠸ࡎࢀࡶ DMSO
stock solution ࢆ࢔ࢵࢭ࢖┤๓࡟ㄪ〇ࡍࡿࡇ࡜࡜ࡍࡿ㸬

2.5

ჾල࣭ᶵჾ

2.5.1

Solar simulator ග⥺㐣ᩄ⑕ࡢ࡯࡜ࢇ࡝ࡀኴ㝧ග࡬ࡢ᭚㟢࡟ࡼࡗ࡚ច㉳ࡉࢀࡿࡇ࡜ࢆ⪃៖ࡍࢀࡤ㸪ROS ࢔ࢵࢭ࢖࡟౑⏝ࡍࡿ solar simulator ࡣྍ⬟࡞㝈ࡾ CIE85/1989 ᶆ‽᫨ග࡟㏆࠸ࡇ࡜ࡀᮃࡲࡋ
࠸㸬౛࠼ࡤ㸪ග※࡜ࡋ࡚ Xe ࣛࣥࣉ (1,500 W) ࢆ⿦ഛࡋࡓ Atlas Suntest CPS/CPS plus (Atlas
Material Technology LLC, Chicago, USA) ࠶ࡿ࠸ࡣ Seric SXL-2500V2 (SERIC., Ltd., Tokyo,
Japan) ࢆ౑⏝ࡋ㸪ᶆ‽᫨ග࡟ࡼࡾ㏆ࡃࡍࡿ࡭ࡃ 290 nm ௨ୗࡢ↷ᑕගࢆ࢝ࢵࢺࡍࡿࡓࡵ࡟
UV ࣇ࢕ࣝࢱ࣮ࢆ⿦╔ࡋ࡚⏝࠸ࡿ㸬UVA detector Dr. Hönle #0037 (Dr. Hönle, München,
Germany) ࡛↷ᑕගࢆࣕࣥࣂ࣮ෆࡢ

ᐃࡋࡓ࡜ࡁ㸪⣙ 2 mW/cm2 ࡢ↷ᑕᙉᗘ࡛࠶ࡾ㸪solar simulator ࡢࢳᗘࡣ 20–29Υ ࡢ⠊ᅖ࡟ไᚚࡉࢀ࡚࠸ࡿࡇ࡜ࡀᮃࡲࡋ࠸㸬ࡲࡓ㸪solar simulator࡟ࡼࡗ࡚ࡣ↷ᑕ࣒ࣛࡀⓎ⏕ࡍࡿࡇ࡜ࡀ࠶ࡿࡢ࡛㸪࢔ࢵࢭ࢖ࡢ๓࡟᳨ドࡋ㸪↷ᑕ࣒ࣛࡀ࡞࠸
㡿ᇦࢆ᫂☜࡟ࡋ࡚࠾ࡃᚲせࡀ࠶ࡿ㸬ᮏ✏࡛ᥦ♧ࡍࡿࢹ࣮ࢱࡣࡍ࡭࡚ࡇࢀࡽࡢ↷ᑕ⿦⨨ࢆ⏝࠸࡚ ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡋࡓࡶࡢ࡛࠶ࡿ㸬௚ࡢ solar simulator ࢆ⏝࠸࡚౑⏝ࡍࡿࡇ࡜ࡶྍ
⬟࡛࠶ࡿࡀ㸪ࡑࡢሙྜ࡟ࡣ㝧ᛶᑐ↷㸦quinine㸧㸪㝜ᛶᑐ↷㸦sulisobenzone㸧࡞ࡽࡧ࡟᥎ዡ໬
ྜ≀ࢭࢵࢺ㸦recommended chemical set㸧ࢆ⏝࠸࡚᥎ዡࣉࣟࢺࢥ࣮ࣝ࡟グ㍕ࡉࢀ࡚࠸ࡿ᮲௳᭱㐺໬ࢆᚲせ࡜ࡍࡿ㸬

2.5.2

ROS࢔ࢵࢭ࢖⏝reaction container ᨃఝኴ㝧ගࢆ↷ᑕࡍࡿ㝿࡟ࡣࢧࣥࣉࣝࢆ 96 well microplate (ᖹᗏ㸪ࢥ࣮ࢸ࢕ࣥࢢ࡞ࡋ) ࡟ධࢀ࡚⾜࠺ࡀ㸪ࡑࡢ㝿࡟ࡣỈศ᥹Ⓨ࡟ࡼࡿࢧࣥࣉࣝ⃰ᗘኚືࢆᅇ㑊ࡍࡿࡓࡵ࡟ࣇࢱࢆࡍࡿ
ࡇ࡜ࡀᚲせ࡛࠶ࡿ㸬㏻ᖖࡢ microplate ࡢࣇࢱ࡛ࡣ↷ᑕගࡢࢫ࣌ࢡࢺࣝࣃࢱ࣮ࣥࡀኚ໬ࡍࡿ
ྍ⬟ᛶࡀ㧗ࡃ㸪ࡑࢀᨾ solar simulator ࠿ࡽࡢ↷ᑕග࡟ᙳ㡪ࡀฟ࡞࠸ࡼ࠺␃ពࡍ࡭ࡁ࡛࠶ࡿ㸬ᮃࡲࡋࡃࡣᅗ 2 㸦ḟ㡫㸧࡟♧ࡍ quartz reaction container (࢜ࢨ࣡⛉Ꮫ㸪ឡ▱) ࢆ⏝࠸ࡿࡇ࡜ࡀ᥎ዡࡉࢀࡿࡀ㸪ࡇࢀ࡟㝈ᐃࡉࢀࡿࡶࡢ࡛ࡣ࡞ࡃྠ➼ရࢆ⏝࠸࡚ࡶࡼ࠸㸬᥎ዡࡉࢀࡿ quartz
reaction container ࡣࢫࢳ࣮ࣝ〇ࡢᅛᐃල㸪ⓑⰍࢸࣇࣟࣥࢩ࣮ࢺ㸪ࡑࡋ࡚ࣇࢱࡢᙺ┠ࢆᯝࡓࡍquartz plate ࠿ࡽᵓᡂࡉࢀࡿ㸬ࡲࡓ㸪Onoue ࡽࡣඛ࡟㸪96 well ࡢ࠺ࡕᅗ 2 ࡟♧ࡍ㡿ᇦ (B2–
G11) ࡟ࡣ࡯ࡰྠ➼ࡢ↷ᑕ㔞ࡢᨃఝኴ㝧ගࡀὀࡀࢀࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋ࡚࠾ࡾ (Onoue et al.,13
19

2008)㸪ࡑࢀᨾ㸪ಙ㢗࠶ࡿࢹ࣮ࢱࢆᚓࡿࡓࡵ࡟ࡣࡇࡢ㡿ᇦࢆ⏝࠸࡚ ROS ࢔ࢵࢭ࢖ࢆ⾜࠺ࡇ࡜ࡀ᥎ዡࡉࢀࡿ㸬

2.5.3

ࡑࡢ௚࡟ᚲせ࡜ࡉࢀࡿჾල࣭⿦⨨ ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡍࡿ㝿࡟ࡣඛ㏙ࡢ⿦⨨࣭ჾලࡢ࡯࠿㸪ᰯṇࡉࢀࡓ UVA detector㸪ᗘィ㸪࣐࢖ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮㸪ගᏛ㢧ᚤ㙾㸪࣎ࣝࢸࢵࢡࢫ࣑࢟ࢧ࣮㸪ࣉ࣮ࣞࢺࢩ࢙࣮
࣮࢝㸪㉸㡢ἼὙίᶵ㸪࣐࢖ࢡࣟࣆ࣌ࢵࢺ࡞࡝ࡀᚲせ࡛࠶ࡿ㸬

ᅗ 2

2.6

reaction container ࡢᵓᡂ

ヨ㦂᪉ἲ࡞ࡽࡧ࡟ࢹ࣮ࢱゎᯒ ROS ࢔ࢵࢭ࢖࡛ࡣ singlet oxygen ࡜ superoxide ࡢ཮᪉ࢆศᯒࡍࡿࡓࡵ㸪ࡲࡎࡑࢀࡒࢀࡢᐃ࡟ᚲせ࡞ assay mixture ࢆᅗ 3 ࡟ᚑ࠸సᡂࡍࡿ㸬singlet oxygenᐃ⏝ࡢ assay mixture(1 mL) ࡟ࡣ௵ពࡢ⃰ᗘࡢ⿕㦂≀㉁㸪50 n mol ࡢ p-nitrosodimethyl aniline㸪50 n mol ࡢ
imidazole ࡀࡑࢀࡒࢀྵࡲࢀ㸪superoxideᐃ⏝ࡢ assay mixture (1 mL) ࡣ௵ពࡢ⃰ᗘࡢ⿕㦂≀㉁࡜ 50 n mol ࡢ NBT ࢆྵ᭷ࡍࡿ㸬ㄪ〇ᚋ㸪200 μL ࡢ assay mixture ࢆ 96 well
microplate ࡟ n=3 ࡟࡞ࡿࡼ࠺ᅗ 4 ࡟ᚑࡗ࡚⛣ࡋ㸪ࡑࡢᚋ࡟ගᏛ㢧ᚤ㙾ほᐹ (100) ࡟ࡼࡗ࡚୙⁐ᛶ⢏Ꮚ࠶ࡿ࠸ࡣᯒฟ≀ࡢ᭷↓ࢆ☜ㄆࡍࡿ㸬ࡶࡋ㸪⿕㦂≀㉁ࡢ⁐ゎᗘࡢၥ㢟࡛᏶඲࡟⁐ゎࡋ࡚࠸࡞࠿ࡗࡓሙྜ࡟ࡣ DMSO stock solution ࢆᕼ㔘ࡋ࡚ 200μM ࡼࡾప࠸⃰ᗘ࡛ࡢ
ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡍࡿࡇ࡜࡛ᑐᛂࡍࡿ㸬㝧ᛶ࡞ࡽࡧ࡟㝜ᛶࢥࣥࢺ࣮ࣟࣝ࡜ࡋ࡚ quinine

1420

(200 μM) ࡜ sulisobenzone (200 μM) ࡢ assay mixture ࢆㄪ〇ࡍࡿ࡜࡜ࡶ࡟㸪blank ࡜ࡋ࡚⿕㦂≀㉁ࡢ stock solution ࡢ௦ࢃࡾ࡟ NaPB ࢆຍ࠼ࡓࡶࡢࡶేࡏ࡚⏝ពࡍࡿ㸬Plate shaker ࡟ࡼࡗ࡚ 5 ⛊⛬ᗘ᧠ᢾࡋࡓᚋ㸪ᨃఝኴ㝧ග↷ᑕ๓ࡢྛ assay mixture ࡢ྾ග್ (singlet oxygenࡣ 440 nm, superoxide ࡣ 560 nm) ࢆࣉ࣮ࣞࢺ࣮ࣜࢲ࣮࡛

ᐃࡍࡿ㸬96 well microplate ࢆquartz reaction container ࡟ࢭࢵࢺࡋ㸪Ẽᐦᛶࢆ㧗ࡵࡿࡓࡵ࡟ quartz plate ࢆࡋࡗ࠿ࡾ࡜ᅛᐃ
ࡍࡿ㸬Solar simulator ࡢࢳࣕࣥࣂ࣮ෆ࡟ assay mixture ࢆධࢀࡿ࠿㸪↷ᑕ࢚ࣜ࢔࡟ assay
mixture ࢆ㓄⨨ࡋ㸪1 ᫬㛫ᨃఝኴ㝧ගࢆ↷ᑕࡍࡿ㸦↷ᑕ㔞㸸Suntest CPS/CPS+ࡣ⣙ 2.0 mW/cm2㸪
SXL-2500V2 ࡣ 3.0̽5.0 mW/cm2㸧㸬㟢ගᚋ࡟ࡣ⿕㦂≀㉁ࡸ NBT+ ࡀᯒฟࡋ࡚࠸ࡿࡇ࡜ࡀ࠶
ࡿࡓࡵ㸪 1 ศ⛬ᗘ plate shaker ࡛ΰ࿴ࡋ㸪ࡑࡢᚋ࡟ࣉ࣮ࣞࢺ࣮ࣜࢲ࣮࡟࡚ྛ assay mixtureࡢ྾ග್ࢆྠᵝ࡟

ᐃࡍࡿ㸬

15
21

ᅗ 3

ROS ࢔ࢵࢭ࢖ࡢ࣮࣡ࢡࣇ࣮ࣟ

Blank ࡞ࡽࡧ࡟⿕㦂≀㉁ࡢ㟢ග๓ᚋࡢ྾ග್࠿ࡽ singlet oxygen ࡞ࡽࡧ࡟ superoxide ࡢ⏘⏕ࢆ௨ୗࡢ㏻ࡾࡑࢀࡒࢀィ⟬ࡍࡿ㸬⿕㦂≀㉁࡟╔Ⰽࡀㄆࡵࡽࢀ㸪࡞࠾࠿ࡘග↷ᑕ࡟ࡼࡗ࡚ኚⰍࡀㄆࡵࡽࢀࡿሙྜ࡟ࡣ㸪ROS ཯ᛂᇶ㉁ࢆྵࡲ࡞࠸ Assay mixture ࢆ⏝ពࡋ࡚ ROS

16
22

assay ࡜ྠᵝࡢ᧯సࢆ⾜࠸㸪440 nm ࠶ࡿ࠸ࡣ 560 nm ࡟࠾ࡅࡿ྾ග್ኚ໬ࢆ ROS assay ࢹ࣮ࢱ࠿ࡽᕪࡋᘬࡃࡇ࡜࡛ᑐᛂࡀྍ⬟࡛࠶ࡿ㸬
Singlet oxygen ࡢ⏘⏕㸸'A440×103 = {A440(-) – A440(+) – (A – B)}103
A440(-): 㟢ග๓ࡢ assay mixture ࡢ 440 nm ࡟࠾ࡅࡿ྾ග್
A440(+): 㟢ගᚋࡢ assay mixture ࡢ 440 nm ࡟࠾ࡅࡿ྾ග್
A:

㟢ග๓ࡢ blank ࡢ 440 nm ࡟࠾ࡅࡿ྾ග್

B:

㟢ගᚋࡢ blank ࡢ 440 nm ࡟࠾ࡅࡿ྾ග್

Superoxide ࡢ⏘⏕㸸'A560×103 = {A560(+) – A560(-) – (B – A)}103
A560(-): 㟢ග๓ࡢ assay mixture ࡢ 560 nm ࡟࠾ࡅࡿ྾ග್
A560(+): 㟢ගᚋࡢ assay mixture ࡢ 560 nm ࡟࠾ࡅࡿ྾ග್

ᅗ 4

2.7

A:

㟢ග๓ࡢ blank ࡢ 560 nm ࡟࠾ࡅࡿ྾ග್

B:

㟢ගᚋࡢ blank ࡢ 560 nm ࡟࠾ࡅࡿ྾ග್

ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿࢧࣥࣉࣝࣞ࢖࢔࢘ࢺ

ࢹ࣮ࢱ᥇⏝᮲௳

 ROS ࢔ࢵࢭ࢖ࢆ⾜࠺㝿࡟ࡣ௨ୗࡢ᮲௳ࢆ‶ࡓࡋࡓሙྜ࡟ࡢࡳࢹ࣮ࢱࢆ᥇⏝ࡍࡿࡶࡢ࡜ࡍ
ࡿ㸬

17
23

(i) 㟢ග๓ࡢ assay mixture ୰࡟࠾࠸࡚⿕㦂≀㉁ࡀ⁐ゎࡋ࡚࠸ࡿࡇ࡜
(ii) blank㸪㝧ᛶ࡞ࡽࡧ࡟㝜ᛶᶆ‽≀㉁࡟ࡘ࠸࡚ࡶྠ᫬࡟ ROS ࢔ࢵࢭ࢖࡛ホ౯ࡋ࡚࠸ࡿࡇ࡜㸬ࡑࡢ㝿࡟ࡣ㝧ᛶᶆ‽≀㉁ quinine (200 μM) ࡢ singlet oxygen ࢹ࣮ࢱࡀ 300 ௨ୖ㸪superoxide
ࡀ 200 ௨ୖ࡛࠶ࡾ㸪㝜ᛶᶆ‽≀㉁ sulisobenzone (200 μM) ࡢ singlet oxygen ࢹ࣮ࢱࡀ 25 ௨ୗ㸪superoxide ࡀ 20 ௨ୗ࡛࠶ࡿࡇ࡜㸬(iii) ᶆ‽≀㉁࡞ࡽࡧ࡟⿕㦂≀㉁ࡢ assay mixture ࡢ 440 nm ࠶ࡿ࠸ࡣ 560 nm ࡟࠾ࡅࡿ྾
ග್ࡀ 0.02 ࠿ࡽ 1.5 ࡢ⠊ᅖ࡟ධࡗ࡚࠸ࡿࡇ࡜㸬

2.8

㝧ᛶᇶ‽

 ⣙ 40 ✀ࡢᕷ㈍་⸆ရ໬ྜ≀࡟࠾ࡅࡿ ROS ࢔ࢵࢭ࢖ࢹ࣮ࢱ࡜ࡑࢀࡽࡢගᏳ඲ᛶ࡟㛵ࡍࡿ⮫ᗋ᝟ሗ࠿ࡽ㸪ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿ⤒㦂๎ⓗ࡞㜈್ࢆタᐃࡋࡓ (Onoue et al., 2008)㸬
⿕㦂≀㉁ (200 μM) ࡢ ROS data ࡟ࡘ࠸࡚㸪singlet oxygen ('A440 nm࣭103): 25 ௨ୖ㸪ࡑࡋ࡚superoxide ('A560 nm࣭103): 20 ௨ୖࡢ࠸ࡎࢀ࠿ࡢ᮲௳ࢆ‶ࡓࡋࡓ࡜ࡁ㸪⿕㦂≀㉁ࡣගឤཷᛶ
ࡀ㧗ࡃ࡞ࢇࡽ࠿ࡢගẘᛶ཯ᛂࢆច㉳ࡍࡿྍ⬟ᛶࡀ࠶ࡿࡶࡢ࡜ุ᩿࡛ࡁࡿ㸬ࡓࡔࡋ㸪⁐ゎᗘࡢၥ㢟࡛ᕼ㔘ୗ࡛ࡢ ROS ࢔ࢵࢭ࢖ࢆవ൤࡞ࡃࡉࢀࡓ㝿࡟ࡣࡇࡢ㝈ࡾ࡛ࡣ࡞࠸㸬ࡍ࡞ࢃࡕ㸪
assay mixture ୰ࡢ⿕㦂≀㉁⃰ᗘࡀ 200 μM ࡼࡾࡶప࠸ሙྜ㸪ROS data ࡀୖグ㜈್ࢆ㉸࠼࡚࠸ࡓሙྜ࡟ࡣ㝧ᛶุᐃࢆ⾜࠺ࡇ࡜ࡀ࡛ࡁࡿࡀ㸪㜈್ࡼࡾࡶప࠸ሙྜ࡟ࡣ㝜ᛶุᐃࡣ࡛ࡁ࡞࠸㸬

3 ヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥ
3.1 ⏝࠸ࡽࢀࡓ≀㉁࡜ࡑࡢጇᙜᛶ
ROS ࢔ࢵࢭ࢖ࡢከ᪋タࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡣ 2 ✀㢮ࡢࢯ࣮࣮ࣛࢩ࣑࣮ࣗࣞࢱࡀ౑⏝ࡉࢀ㸪ࡑࢀࡒࢀ 1 ヨ㦂ࡎࡘィ 2 ヨ㦂ࡀᐇ᪋ࡉࢀࡓ㸬ྛヨ㦂ࡣᢏ⾡⛣㌿ᛶࡢホ౯ࢆ┠ⓗ࡜ࡋ࡚ࣉࣞ
ࣂࣜࢹ࣮ࢩࣙࣥ㸦Phase I㸧ヨ㦂࡜ヨ㦂ἲࡢホ౯ࢆ┠ⓗ࡜ࡍࡿࣂࣜࢹ࣮ࢩࣙࣥ㸦Phase II㸧ࡼࡾᵓᡂࡉࢀࡓ㸬ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ㸪ࣂࣜࢹ࣮ࢩࣙࣥ࡟ඹ㏻ࡋ࡚㸪㝧ᛶᑐ↷≀㉁࡜ࡋ࡚
quinine HCl ࢆ㸪㝜ᛶᑐ↷≀㉁࡜ࡋ࡚ sulisobenzone ࡀ౑⏝ࡉࢀࡓ㸬Quinine HCl ࡣࣄࢺࡢගẘᛶ≀㉁࡜ࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿ㸦Ljunggren et al, 1986㸧㸬Sulisobenzone ࡣ 3T3 NRU-PT ࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ࣄࢺࢹ࣮ࢱࢆࡶ࡜࡟㠀ගẘᛶ≀㉁࡟ศ㢮ࡉࢀ࡚࠸ࡿ(Spielmann et al,
1998㸧㸬
࡞࠾㸪ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡓ 5-fluorouracil (5-FU)ࡣࣄࢺ࡛ගẘᛶ≀㉁࡜ࡋ࡚ሗ࿌
ࡉࢀ࡚࠸ࡿ㸦Dillaha et al, 1983㸧ࡀ㸪3T3 NRU-PT ࡛ࡣගẘᛶࢆ♧ࡉ࡞࠸࡜ሗ࿌ࡉࢀ࡚࠸ࡿ

18
24

㸦Kleinman et al, 2010 and Onoue et al, 2010㸧㸬5-FU ࡟ࡼࡿගẘᛶࡢཎᅉ࡜ࡋ࡚㸪UV-B ࡢࡳ࡟཯ᛂࡋ࡚࠸ࡿ࡜ࡢሗ࿌㸦Kirkup M.E. et al, 2003 and Andersen K.E. et al, 1984㸧ࡸ UV-B ࡛ගศゎࡍࡿ㝿ࡢ ROS ⏕ᡂ࡟ࡼࡿ࡜ࡢሗ࿌㸦Miolo G. et al, 2011㸧ࡀ࠶ࡿ㸬5-FU ࡣ⮬↛ࡢኴ㝧ගࡢἼ㛗⠊ᅖ࡛ࡣ୺࡟ UV-B㸦290̽320 nm㸧ࢆ྾཰ࡍࡿ㸦ࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩㸪Appendix 7㸧ࡇ࡜࠿ࡽ㸪5-FU ࡢග໬Ꮫⓗ࡞άᛶ໬࡟ࡣ UV-B ࡢ↷ᑕࡀᚲ㡲࡜⪃࠼ࡽࢀࡿ㸬5-FU ࡢගẘᛶ࡟㛵ࡋ࡚㸪㧗ရ㉁࡞ࣄࢺ࡛ࡢࢹ࣮ࢱࡣ࡞ࡃ㸪5-FU ࡀගẘᛶ≀㉁࡛࠶ࡿ࠿࡝࠺࠿ࡣุ᩿࡛ࡁ
࡞࠿ࡗࡓ㸬5-FU ࡣ㸪3T3 NRU-PT ࡔࡅ࡛࡞ࡃ㸪ROS ࢔ࢵࢭ࢖࡛ࡶ㝧ᛶ⤖ᯝࢆ♧ࡉ࡞࠸࡜ࡢ
᝟ሗࡀ࠶ࡗࡓࡇ࡜࠿ࡽ㸪ROS ࢔ࢵࢭ࢖ࡢ㝈⏺࡟㛵ࡍࡿ᝟ሗࢆ☜ㄆࡍࡿࡓࡵ࡟ࣉࣞࣂࣜࢹ࣮
ࢩࣙࣥ⏝ࡢ໬ྜ≀࡜ࡋ࡚㑅ᢥࡉࢀࡓ㸬୍᪉㸪5-FU ࢆྵࡴࣇࢵ໬ࣆ࣑ࣜࢪࣥ⣔⸆๣ࡢ๪స⏝࡜ࡋ࡚ᡭ㊊⑕ೃ⩌㸦㔜⠜๪స⏝⑌ᝈูᑐᛂ࣐ࢽࣗ࢔ࣝ㸪ᡭ㊊⑕ೃ⩌㸪ཌ⏕ປാ┬㸪2010㸧ࡀ▱ࡽࢀ࡚࠾ࡾ㸪5-FU ࡢගẘᛶ࡜ࡋ࡚␲ࢃࢀ࡚࠸ࡿ㸬ࡋ࠿ࡋ㸪࠸ࢃࡺࡿ໬Ꮫ≀㉁࡟ࡼࡿ┤᥋ⓗ࡞ගẘᛶ࡟ࡼࡿࡶࡢ࡛ࡣ࡞ࡃ㸪ᡭ㊊⑕ೃ⩌ࡢ⑕≧ࡀኴ㝧ග⥺࡬ࡢ᭚㟢࡟ࡼࡗ࡚ᝏ໬ࡋ
ࡓ⑕౛ࡀྵࡲࢀ࡚࠸ࡿྍ⬟ᛶࡶ⪃៖ࡍ࡭ࡁ࠿ࡶࡋࢀ࡞࠸㸬

3.1.1

ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ

ࣉࣞࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡣ㝜ᛶᑐ↷≀㉁㸪㝧ᛶᑐ↷≀㉁ࢆྵࡴ 13 ≀㉁ࡀ౑⏝ࡉࢀࡓ㸬ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ࡟౑⏝ࡋࡓ≀㉁࡟㛵ࡍࡿ᝟ሗࢆࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࡼࡾᢤ⢋ࡋ㸪Appendix
2 ࠾ࡼࡧ Appendix 3 ࡟♧ࡍ㸬
௚ࡢ 10 ≀㉁ࡢ࠺ࡕ㸪8-MOP㸪amiodarone HCl㸪chlorpromazine㸪doxycycline HCl㸪furosemide㸪
ketoprofen ࠾ࡼࡧ norfloxacin ࡣ 3T3NRU-PT ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢගẘᛶ≀㉁ࡢࣜࢫࢺ࠿ࡽ
㑅ᢥࡉࢀࡓ㸬ࣄࢺࡢࢹ࣮ࢱࡣ 3T3NRU-PT ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟グ㍕ࡉࢀ࡚࠸ࡿ㸦Spielmann
et al, 1994a and 1998a㸧㸬Diclofenac㸪levofloxacin㸪omeprazole ࡢࣄࢺගẘᛶ≀㉁࡜ࡢሗ࿌ࢆ
᰿ᣐ࡟㑅ᢥࡉࢀࡓ㸦Przybilla et al, 1987㸪Boccumini et al, 2000㸪 Dam et al, 2008㸧㸬

3.1.2

ࣂࣜࢹ࣮ࢩࣙࣥ

ࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡓ໬Ꮫ≀㉁ࡢᵓᡂࢆ⾲ 2 ࡟♧ࡍ㸬ගẘᛶ≀㉁ 23 ≀㉁㸪㠀ගẘᛶ≀㉁ 19 ≀㉁ࡢィ 42 ≀㉁ࢆࢥ࣮ࢻ໬ࡋࡓ≧ែ࡛㸪ヨ㦂᪋タ 7 ᪋タ࡟ศ㓄ࡋࡓ㸬42 ≀㉁ࡢᵓᡂ
ࡣ་⸆ရ 23 ≀㉁㸪໬Ꮫ≀㉁ 9 ≀㉁㸪⣸እ⥺྾཰๣ 10 ≀㉁࡛࠶ࡗࡓ㸬ࣂࣜࢹ࣮ࢩࣙࣥ࡟౑
⏝ࡋࡓ≀㉁ࡢ㑅ᢥ᰿ᣐࢆ Appendix 4 ࡟♧ࡋࡓ㸬

1925

⾲ 2 ࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡽࢀࡓ≀㉁ศ㢮ගẘᛶ≀㉁㸦23 ≀㉁㸧

㠀ගẘᛶ≀㉁㸦19 ≀㉁㸧

་⸆ရ
㸦23 ≀㉁㸧

㸦18 ≀㉁㸧
Acridine
Acridine HCl
Amiodarone HCl
Chlorpromazine HCl
Doxycycline HCl
Fenofibrate
Furosemide
Ketoprofen
6-Methylcoumarine໬Ꮫ≀㉁㸦9 ≀㉁㸧

㸦4 ≀㉁㸧
Anthracene
Bithionol
Hexachlorophene
Rose Bengal

㸦5 ≀㉁㸧
Sodium dodecyl sulphateCinnamic acid
Chlorhexidine
L-histidine
p-Aminobenzoic acid⣸እ⥺྾཰๣
㸦10 ≀㉁㸧

㸦1 ≀㉁㸧
Butyl methoxydibenzoylmethane (Avobenzone)

㸦9 ≀㉁㸧
Benzylindene camphor sulphonic acid
4-Methyl benzylidene camphor
Octyl salicylate
Octyl methoxycinnamate
2-Ethylhexyl 4-methoxycinnamate
2-(2-Hydroxy-5-methylphenyl)benzotriazole
2-tert-Butyl-6-(5-chloro-2H-benzotriazol-2-yl)-4-methylphenol
2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramet
hylbutyl)phenol
2-(2H-Benzotriazol-2-yl)-6-dodecyl-4-methylphenol

8-Methoxy psoralen
Nalidixic acid
Nalidixic acid (Nasalt)
Norfloxacin
Ofloxacin
Piroxicam
Promethazine HCl
Rosiglitazone
Tetracycline㸦5 ≀㉁㸧
AspirinBenzocaine
Erythromycin
Phenytoin
Penicilin G

3.1.3 ヨ㦂ἲࡢṇ☜ᛶࢆホ౯ࡍࡿࡓࡵ࡟⏝࠸ࡽࢀࡓ໬Ꮫ≀㉁ࡢ invivo ཧ↷ࢹ࣮ࢱ
ROS ࢔ࢵࢭ࢖ࡢከ᪋タࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ࡣ㸪ࢥ࣮ࢻ໬ࡋࡓ໬ྜ≀ࢆ⏝࠸࡚ᐇ᪋ࡋࡓ
ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢ⤖ᯝࢆ⏝࠸࡚ヨ㦂ἲࡢṇ☜ᛶࢆホ౯ࡋࡓ㸬ࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡓ 42 ≀㉁ࡢග໬Ꮫⓗ≉ᛶ࠾ࡼࡧࣄࢺ㸪ື≀㸪in vitro ホ౯⣔࡛ࡢගẘᛶ࡟㛵ࡍࡿ᝟ሗࢆࣂ
ࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࡼࡾᢤ⢋ࡋ㸪Appendix 5 ࡟♧ࡋࡓ㸬౑⏝ࡋࡓ≀㉁ࡢ࠺ࡕ㸪ගẘᛶ≀㉁࡜
㠀ගẘᛶ≀㉁ࡀ࡯ࡰ༙ࠎ࡛࠶ࡿࡇ࡜ࡣឤᗘࡸ≉␗ᗘ➼㸪ヨ㦂ἲࡢṇ☜ᛶࢆホ౯ࡍࡿࡓࡵ࡟
ᐇ᪋ࡍࡿࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟㐺ࡋ࡚࠸ࡓ࡜ᛮࢃࢀࡿ㸬ࡋ࠿ࡋ࡞ࡀࡽ㸪་⸆ရ࡛ࡣගẘᛶ≀㉁ࡢ๭ྜࡀከࡃ㸪⣸እ⥺྾཰๣࡛ࡣ㠀ගẘᛶ≀㉁ࡢ๭ྜࡀከࡃ㸪≀㉁ࡢ✀㢮࡟ࡼࡗ࡚㸪㑅ᐃࡉࢀࡓ㝧ᛶ≀㉁࡜㝜ᛶ≀㉁࡟೫ࡾࡀ࠶ࡗࡓ㸬

20
26

࡞࠾㸪➨୕⪅ホ౯ጤဨ఍ࡣࡑࡢሗ࿌᭩࡟࠾࠸࡚㸪౑⏝ࡋࡓ໬Ꮫ≀㉁࡟㛵ࡋࠕ➨୕⪅ホ౯ጤဨ఍ࡣࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛㑅ᢥࡉࢀࡓ໬Ꮫ≀㉁ࡣ࢔ࢵࢭ࢖ࢆホ౯ࡍࡿ໬Ꮫ≀㉁ࡢ௦⾲࡜ࡋ࡚㐺ษ࠿ࡘ༑ศ࡞࡛࠶ࡿࡇ࡜࡟ྠពࡋࡓ㸬ࣄࢺගẘᛶ≀㉁ࢆྵࡴ 42 ≀㉁ࡣ㝧ᛶ࡜ࡋ࡚▱ࡽࢀࡿ 23 ≀㉁㸪㝜ᛶ 19 ≀㉁ࡼࡾᵓᡂࡉࢀࡓ㸬ࡑࢀࡽࡢ≀㉁ࡣࣄࢺࣃࢵࢳࢸࢫࢺ࡜ 3T3
NRU-PT ࡢࢹ࣮ࢱ࡟⿬௜ࡅࡽࢀ࡚࠸ࡓ㸬ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ᚓࡽࢀࡓࡍ࡭࡚ࡢࢹ࣮ࢱࡣࣂ
ࣜࢹ࣮ࢩࣙࣥヨ㦂ሗ࿌᭩࡟グ㍕ࡉࢀ࡚࠸ࡿ㸬ࠖ࡜グ㍕ࡋ࡚࠸ࡿ㸬

3.2 ࣂࣜࢹ࣮ࢩࣙࣥ⤖ᯝࡢホ౯
3.2.1 ヨ㦂ἲࡢࢹ࣮ࢱ࡜⤖ᯝࡢ฼⏝ᛶ
ROS ࢔ࢵࢭ࢖ከ᪋タࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡣ 2 ✀㢮ࡢࢯ࣮࣮ࣛࢩ࣑࣮ࣗࣞࢱࡀ౑⏝ࡉࢀ㸪2
ヨ㦂ᐇ᪋ࡉࢀࡓ㸬1 ヨ㦂┠ࡣ࢔ࢺࣛࢫ♫ࡢ Suntest CPS/CPS+ࢆ⏝࠸㸪2011 ᖺ 3 ᭶㹼10 ᭶࡟࠿ࡅ࡚㸪3 ᪋タ࡛ᐇ᪋ࡉࢀ㸪ࡶ࠺ 1 ヨ㦂ࡣࢭࣜࢵࢡ♫ࡢ SXL-2500V2 ࢆ⏝࠸㸪2011 ᖺ 5 ᭶㹼2012 ᖺ 6 ᭶࡟࠿ࡅ࡚ 4 ᪋タ࡛ᐇ᪋ࡉࢀࡓ㸬ࡇࢀࡽࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢࢹ࣮ࢱ࡜⤖ᯝࡣࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩୰࡟グ㍕ࡉࢀ㸪฼⏝ྍ⬟࡛࠶ࡿ㸬

3.2.2 ヨ㦂᪉ἲࡢṇ☜ᛶࣂࣜࢹ࣮ࢩࣙࣥヨ㦂⤖ᯝ࠿ࡽ⟬ฟࡋࡓヨ㦂᪋タࡢឤᗘ㸪≉␗ᗘ㸪㝧ᛶண⋡㸪㝜ᛶண⋡࠾ࡼࡧṇ☜ᛶࢆ⾲ 3 ࡟♧ࡋࡓ㸬㞴⁐ᛶࡢࡓࡵホ౯୙ྍ⬟࡛࠶ࡗࡓ≀㉁ࢆ㝖ࡃ࡜㸪ගẘᛶ≀㉁࡟ᑐࡍࡿឤᗘࡣ඲᪋タ࡜ࡶ࡟ 100%࡛࠶ࡾ㸪㝜ᛶண⋡ࡶྛ᪋タ࡜ࡶ 100%࡛࠶ࡾ㸪ഇ㝜ᛶࡣ࡞࠿ࡗࡓ㸬21
27

⾲ 3 ROS ࢔ࢵࢭ࢖ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂⤖ᯝ
ヨ㦂
᪋タ

ឤᗘ

≉␗ᗘ

㝧ᛶ
ண ⋡

㝜ᛶ
ண ⋡

ṇ☜ᛶ

100%
(21/21)75.0%(12/16)84.0%(21/25)100%(12/12)

89.2%(33/37)100%(18/18)71.4%(10/14)

81.8%(18/22)100%(10/10)87.5%(28/32)

3

100%(20/20)88.2%(15/17)90.9%(20/22)100%(15/15)

94.6%(35/37)4

100%(19/19)71.4%(10/14)

82.6%(19/23)100%(10/10)87.9%(29/33)5

100%(19/19)50.0%(6/12)

76.0%(19/25)

100%
(6/6)

80.6%
(25/31)100%(19/19)73.3%(11/15)

82.6%(19/23)100%(11/11)

88.2%(30/34)100%(18/18)69.2%(9/13)

81.8%(18/22)

100%
(9/9)

87.1%
(27/31)ேᕤኴ㝧ⅉග※

1
2

6
7

࢔ࢺࣛࢫ♫
SuntestCPS/CPS+ࢭࣜࢵࢡ♫SXL-2500V2

Weakly photoreactive ࡜ุᐃࡉࢀࡓ≀㉁ࢆගẘᛶ≀㉁࡜ุᐃࡋࡓሙྜࡢุᐃ⤖ᯝࢆ♧ࡍ㸬ឤᗘ㸸ගẘᛶ≀㉁ࡀ ROS ࢔ࢵࢭ࢖࡛㝧ᛶࢆ♧ࡍ๭ྜ㸦㸣㸧≉␗ᗘ㸸㠀ගẘᛶ≀㉁ࡀ ROS ࢔ࢵࢭ࢖࡛㝜ᛶࢆ♧ࡍ๭ྜ㸦㸣㸧㝧ᛶண ⋡㸸ROS ࢔ࢵࢭ࢖࡛㝧ᛶࡔࡗࡓ≀㉁ࡢ࠺ࡕ㸪ගẘᛶ≀㉁ࡢ๭ྜ㸦㸣㸧㝜ᛶண ⋡㸸ROS ࢔ࢵࢭ࢖࡛㝜ᛶࡔࡗࡓ≀㉁ࡢ࠺ࡕ㸪㠀ගẘᛶ≀㉁ࡢ๭ྜ㸦㸣㸧ṇ☜ᛶ㸸ROS ࢔ࢵࢭ࢖ࡢุᐃ⤖ᯝࡀගẘᛶ≀㉁㸪㠀ගẘᛶ≀㉁ࢆṇࡋࡃุᐃࡋࡓ๭ྜ㸦㸣㸧

3.2.3 ヨ㦂ࢹ࣮ࢱࡢ෌⌧ᛶྛ᪋タ࡟࠾ࡅࡿ㝧ᛶᑐ↷≀㉁㸪㝜ᛶᑐ↷≀㉁ࡢ᪥ෆኚືࢆᅗ 5 ࡟㸪᪥㛫ኚືࢆᅗ 6 ࡟♧ࡋ㸪ࡲࡓ㸪ග※⿦⨨㛫ࡢ෌⌧ᛶࢆᅗ 7 ࡟♧ࡋࡓ㸬᪥ෆኚື㸪᪥㛫ኚື࡜ࡶ࡟ᑠࡉࡃ㸪᪋タ㛫㸪␗࡞ࡿග※⿦⨨㛫ࡢኚືࡶᑠࡉ࠿ࡗࡓ㸬ヨ㦂⣔ࡢ᪋タෆ㸪᪋タ㛫ࡢ෌⌧ᛶࡣ㧗ࡃ㸪ග※⿦⨨㛫ࡢỗ⏝ᛶ࡟ၥ㢟࡞࠿ࡗࡓ㸬ࡲࡓ㸪ྛ᪋タ࡟࠾ࡅࡿヨ㦂⤖ᯝࡢ෌⌧ᛶ࡟ࡘ࠸࡚㸪⾲ 4
࡟♧ࡋࡓ㸬

22
28

㻌㻌

㻌

ᅗ5

ྛヨ㦂᪋タ࡟࠾ࡅࡿ㝧ᛶᑐ↷≀㉁㸪㝜ᛶᑐ↷≀㉁ࡢ᪥ෆኚື
᪥ෆኚືࡣྛ᪋タ࡛᭱ࡶከࡃࡢ ᐃࢆᐇ᪋ࡋࡓ᪥ࡢ㝧ᛶ࠾ࡼࡧ㝜ᛶᑐ↷≀㉁ࡢ⤖ᯝ࠿ࡽ⟬ฟࡋࡓ㸬Lab#1–3: Atlas Suntest CPS(⣙ 2.0 mW/cm2)㸪Lab#4–7: Seric SXL-2500V2 (3.0–5.0 mW/cm2).,‫ۍ‬: quinine ࡢ singlet oxygen, ‫ی‬: quinine ࡢ superoxide; ‫ۑ‬: sulisobenzone ࡢ singlet oxygen, ‫ە‬:
sulisobenzone ࡢ superoxide, ࢹ࣮ࢱࡣ meansSD ࢆ♧ࡍ(n=9)㸬

ᅗ6

ྛヨ㦂᪋タ࡟࠾ࡅࡿ㝧ᛶᑐ↷≀㉁㸪㝜ᛶᑐ↷≀㉁ࡢ᪥㛫ኚື᪥㛫ኚືࡣྛ᪋タࡢ ᐃ᪥࡟࠾ࡅࡿ᭱ึࡢ ᐃ࡟࠾ࡅࡿ㝧ᛶ࠾ࡼࡧ㝜ᛶᑐ↷≀㉁ࡢ⤖ᯝ࠿ࡽ⟬ฟࡋࡓ㸬Lab#1–3: Atlas Suntest CPS(⣙ 2.0 mW/cm2)㸪Lab#4–7: Seric SXL-2500V2 (3.0–5.0 mW/cm2).,‫ۍ‬: quinine ࡢ singlet oxygen, ‫ی‬: quinine ࡢ superoxide; ‫ۑ‬: sulisobenzone ࡢ singlet oxygen, ‫ە‬:
sulisobenzone ࡢ superoxide, ࢹ࣮ࢱࡣ meansSD ࢆ♧ࡍ(days 1, 2, and 3; n=9)㸬

㻌

23
29

㻌
ᅗ7

㝧ᛶᑐ↷≀㉁㸪㝜ᛶᑐ↷≀㉁ࡢග※⿦⨨㛫ࡢ෌⌧ᛶ᪥㛫ኚືࡣྛ᪋タࡢ ᐃ᪥࡟࠾ࡅࡿ᭱ึࡢ ᐃ࡟࠾ࡅࡿ㝧ᛶ࠾ࡼࡧ㝜ᛶᑐ↷≀㉁ࡢ⤖ᯝ࠿ࡽ⟬ฟࡋࡓ㸬Lab#1̽3: Atlas Suntest CPS(⣙ 2.0 mW/cm2)㸪Lab#4̽7: Seric SXL-2500V2 (3.0̽5.0 mW/cm2).,‫ۍ‬: quinine ࡢ singlet oxygen, ‫ی‬: quinine ࡢ superoxide; ‫ۑ‬: sulisobenzone ࡢ singlet oxygen, ‫ە‬:
sulisobenzone ࡢ superoxide, ࢹ࣮ࢱࡣ meansSD ࢆ♧ࡍ(days 1, 2, and 3; n=9)㸬

⾲ 4 ࣂࣜࢹ࣮ࢩࣙࣥ⤖ᯝࡢග※⿦⨨㛫㸪᪋タ㛫ࡢ෌⌧ᛶࣂࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡓ໬Ꮫ≀㉁
ගẘᛶ≀㉁

㝧ᛶ
ROS

㠀ගẘᛶ≀㉁Suntest CPS

SXL-2500V2

Suntest CPS

SXL-2500V2[#1] 21[#4] 19[#1] 4[#4] 4[#2] 18[#5] 19[#2] 4[#5] 6[#3] 20[#6] 19[#3] 2[#6] 4[#7] 18

࢔ࢵࢭ࢖[#7] 4ࣂࣜࢹ࣮ࢩࣙ

Suntest CPS

SXL-2500V2

Suntest CPS

SXL-2500V2ࣥ⤖ᯝ[#1] 0[#4] 0[#1] 12[#4] 10[#2] 0[#5] 0[#2] 10[#5] 6[#3] 0[#6] 0[#3] 15[#6] 11㝜ᛶ

[#7] 0[#7] 9

Lab#1̽3: Atlas Suntest CPS(⣙ 2.0 mW/cm2)㸪Lab#4̽7: Seric SXL-2500V2 (3.0̽5.0 mW/cm2)
ᩘ್ࡣ ROS ࢔ࢵࢭ࢖࡛㝧ᛶ࠶ࡿ࠸ࡣ㝜ᛶࢆ♧ࡋࡓࢆ♧ࡋࡓ≀㉁ࡢ࠺ࡕ㸪ගẘᛶ≀㉁࠶ࡿ࠸ࡣ㠀ගẘᛶ≀㉁ࡢಶᩘࢆ♧ࡍ㸬

2430

3.2.4 ヨ㦂᪉ἲࡢࢹ࣮ࢱࡢಙ㢗ᛶࣂࣜࢹ࣮ࢩࣙࣥ࡟ཧຍࡋࡓࡍ࡭࡚ࡢヨ㦂᪋タࡀ᪥ᖖⓗ࡟ GLP 㐺ྜヨ㦂ࢆᐇ᪋ࡋ࡚࠸ࡿ᪋タ࡛ࡣ࡞࠸ࡀ㸪ࢹ࣮ࢱࡢಙ㢗ᛶಖドࡣ࡞ࡉࢀࡓ㸬ࡍ࡞ࢃࡕ㸪ࡍ࡭࡚ࡢ⏕ࢹ࣮ࢱ࠾ࡼࡧゎᯒ⤖ᯝࡣྛ᪋タ࡟࠾࠸࡚☜ㄆࡉࢀ㸪ࡑࡢᚋ㸪ࣂࣜࢹ࣮ࢩࣙࣥ㐠Ⴀဨ఍ࡢಙ㢗ᛶಖドࢢ࣮ࣝࣉ࡛☜ㄆࡉࢀ࡚࠸ࡓ㸬ࡇࢀࡽࡢ᧯సࢆ㏻ࡌ㸪ಙ㢗ᛶಖドࡢほⅬ࡛㸪⏕ࢹ࣮ࢱࡀṇ☜࡟ヨ㦂⤖ᯝ࡟཯ᫎࡉࢀ࡚࠸ࡿࡇ࡜ࡀ☜ㄆࡉࢀ࡚࠸ࡓ㸬

4 ヨ㦂᪉ἲ࡟㛵ࡍࡿ௚ࡢ⛉Ꮫⓗ⪃ᐹ
4.1 ග࡟୙Ᏻᐃ࡞≀㉁࡬ࡢ㐺⏝
ROS ࢔ࢵࢭ࢖ࡣ་⸆ရࡢග໬Ꮫⓗ཯ᛂᛶࢆ㎿㏿࠿ࡘ⡆౽࡟ᐃ࡛ࡁࡿࡀ㸪ࡑࡢ཯㠃㸪ᚓࡽࢀࡿࢹ࣮ࢱ࠿ࡽࡣගᏳ඲ᛶࡔࡅ࡛ࡣ࡞ࡃ㸪ගᏳᐃᛶ࡞࡝࡟ၥ㢟ࡀ࠶ࡿ໬ྜ≀ࡶྵࡵ࡚㝧ᛶ࡜ุูࡍࡿࡇ࡜ࡀ࠶ࡿ㸬ࡇࢀࡣ ROS ࢔ࢵࢭ࢖ࡀගẘᛶ཯ᛂࡢࡁࢃࡵ࡚ึᮇ࡟㉳ࡇࡿග໬Ꮫⓗ཯ᛂᛶࢆホ౯ࡍࡿࢶ࣮࡛ࣝ࠶ࡿࡀᨾࡢㄢ㢟࡛࠶ࡾ㸪ගᏳ඲ᛶホ౯࡟࠾ࡅࡿ୍ḟࢫࢡ࣮ࣜࢽࣥࢢ࡜ࡋ࡚ᮏἲࢆ฼⏝ࡍࡿ㝿࡟ࡣࡇࡢⅬࢆ⇍⪃ࡢୖࢹ࣮ࢱࡢゎ㔘ࢆ⾜࠺ᚲせࡀ࠶ࡿ㸬Onoue ࡽࡣࡇࡢㄢ㢟ࢆゎỴࡍ࡭ࡃ㸪ヨ㦂⁐ᾮ࡜ඹ࡟ᣦ♧⸆ࢆῧຍࡋ࡞࠸ヨ㦂ࢥࣥࢺ࣮ࣟࣝ⁐ᾮࢆㄪ〇ࡋ㸪཯ᛂᚋ࡟཯ᛂ⁐ᾮ࠿ࡽヨ㦂ࢥࣥࢺ࣮ࣟࣝ⁐ᾮ࡛ᚓࡽࢀࡓ್ࢆᕪࡋᘬࡃᡭἲࢆ
ᥦ᱌ࡋ࡚࠾ࡾ㸪ගᏳᐃᛶ࡟ၥ㢟ࡢ࠶ࡿ໬ྜ≀࡟ᑐࡍࡿண

⢭ᗘࡢᨵၿࡀㄆࡵࡽࢀ࡚࠸ࡿ

(Onoue et al., 2013)㸬

4.2 㞴⁐ᛶ≀㉁࡬ࡢᑐᛂ
㏆ᖺࡢ๰⸆άື࡟࠾࠸࡚ࡣỈ࡟㞴⁐ᛶࡢ໬ྜ≀ࡀ་⸆ရೃ⿵≀㉁࡜ࡋ࡚๰ฟࡉࢀࡿࡇ࡜
ࡀ㠀ᖖ࡟ከ࠸ࡀ㸪ROS ࢔ࢵࢭ࢖࡟࠾࠸࡚ࡣᇶᮏⓗ࡟ 200 μM ࡢ⃰ᗘ࡛ࢫࢡ࣮ࣜࢽࣥࢢࢆ⾜࠺ࡓࡵ࡟ assay mixture ୰࡛ࡢᯒฟࢆㄆࡵࡿࡇ࡜ࡀ࠶ࡿ㸬ࡑࡢሙྜ࡟ࡣࡼࡾప⃰ᗘ࡟࡞ࡿࡼ
࠺ᕼ㔘ࢆ⾜ࡗ࡚ ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡍࡿࡇ࡜ࡀྍ⬟࡛ࡣ࠶ࡿࡀ㸪ࡑࡢ㝿࡟ࡣᚓࡽࢀࡓࢹ࣮
ࢱ࡟ࡼࡗ࡚㝧ᛶุᐃࢆฟࡍࡇ࡜ࡣ࡛ࡁ࡚ࡶ㝜ᛶุᐃࢆฟࡍࡇ࡜ࡀࡁࢃࡵ࡚㞴ࡋ࠸㸬Onoueࡽࡣࡇࡢㄢ㢟ࢆゎỴࡍ࡭ࡃ㸪⏺㠃άᛶ๣ࢆ฼⏝ࡋࡓ ROS ࢔ࢵࢭ࢖ࡸ࢔ࣝࣈ࣑ࣥࢆ assay
mixture ࡟ຍ࠼ࡓᨵⰋᆺ ROS ࢔ࢵࢭ࢖ࢆᥦ᱌ࡋ࡚࠾ࡾ㸪ᮏᡭἲ࡛ࡣ㞴⁐ᛶ་⸆ရ࡬ࡢ㐺⏝⋡ࡀ㢧ⴭ࡟ᨵၿࡉࢀࡿ (Onoue et al., 2008; Onoue et al., 2014; Seto et al., 2013)㸬⏺㠃άᛶ๣ࢆ฼⏝ࡋࡓᨵⰋᆺ ROS ࢔ࢵࢭ࢖࡟ࡘ࠸࡚ࡣ㸪㞴Ỉ⁐ᛶࡢ໬⢝ရ⣲ᮦ࡟࠾࠸࡚ࡶࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀ࡚࠾ࡾ㸪ROS ࢔ࢵࢭ࢖ࡢ㐺⏝ᛶᣑ኱࡟኱ࡁࡃᐤ୚ࡍࡿࡶࡢ࡜ᮇᚅࡉࢀࡿ㸦Onoue etal., 2013㸧㸬ࡓࡔࡋ㸪assay mixture ࡢ⤌ᡂࢆኚ࠼ࡓሙྜ࡟ࡣ ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿุᐃᇶ

25
31‽ࡀኚࢃࡿྍ⬟ᛶࡀ࠶ࡾ㸪ㄗࡗࡓගᏳ඲ᛶ᝟ሗࢆᚓࡿࡇ࡜ࡀ࡞࠸ࡼ࠺⣽ᚰࡢὀពࡀᚲせ࡛࠶ࡿ㸬

4.3 ΰྜ≀࠾ࡼࡧศᏊ㔞୙᫂ヨᩱ࡬ࡢᑐᛂ
໬⢝ရ⣲ᮦ࡟࠾࠸࡚ࡣᐃࡲࡗࡓศᏊ㔞ࢆ᭷ࡋ࡚࠸࡞࠸᳜≀ᢳฟ≀➼ࡢΰྜ≀ࡀᩘከࡃᏑ
ᅾࡍࡿࡀ㸪ROS ࢔ࢵࢭ࢖࡟࠾࠸࡚ࡣ㸪๓㏙ࡢ㏻ࡾ 200 ȝM ࡢ⃰ᗘ࡛ࢫࢡ࣮ࣜࢽࣥࢢࢆ⾜࠺ࡓࡵ࡟㸪ࣔࣝ⃰ᗘࡢ⟬ฟ࡛ࡁ࡞࠸ΰྜ≀ࡢヨ㦂ᐇ᪋ࡀ୙ྍ⬟࡛࠶ࡿ㸬Nishida ࡽࡣࡇࡢㄢ㢟ࢆゎỴࡍ࡭ࡃ㸪ヨ㦂⃰ᗘࢆศᏊ㔞ࡀ᫂☜࡛࡞࠸࡜タᐃ࡛ࡁ࡞࠸ࣔࣝ⃰ᗘ࡛ࡣ࡞ࡃ㸪μg/mL༢఩࡛ࡢ ROS ࢔ࢵࢭ࢖ࢆሗ࿌ࡋ࡚࠸ࡿ (Nishida et al., 2015)㸬ᮏሗ࿌࡟౑⏝ࡉࢀࡓ≀㉁ࢆ⾲
5 ࡟♧ࡋ㸪ࡲࡓ㸪ᚓࡽࢀࡓ⤖ᯝࢆ⾲ 6 ࡟♧ࡋࡓ㸬ሗ࿌࡛ࡣ㸪ࡇࢀࡲ࡛࡟ගᏳ඲ᛶୖࡢᠱᛕࡀ
Ꮡᅾࡍࡿ࡜ሗ࿌ࡉࢀ࡚࠸ࡿ໬Ꮫ≀㉁ࡢ࡯࡜ࢇ࡝ࡀศᏊ㔞 500 ௨ୗࡢపศᏊ໬ྜ≀࡛࠶ࡾ㸪ࡑࡢᖹᆒศᏊ㔞ࡣ 250 ࡜௬ᐃ࡛ࡁࡿࡇ࡜㸪ศᏊ㔞 250 ࡢ໬ྜ≀ࡀ 200 ȝM ࡜࡞ࡿ 50 ȝg/mL࡛ࡢヨ㦂࡛㸪ᐃἲࡢ ROS ࢔ࢵࢭ࢖࡜ྠ➼ࡢឤᗘࡀㄆࡵࡽࢀࡿࡇ࡜ࡀ☜ㄆࡉࢀ࡚࠸ࡿ㸬ΰྜ≀ࢆ⏝࠸ࡓ᳨ド࡟࠾࠸࡚ࡶ㸪㐺⏝⃰ᗘ 50 ȝg/mL ࡛ࡢヨ㦂ࡣ㝧ᛶ᳨ฟ⋡ 100%࡛࠶ࡿ᭷⏝࡞ࢫࢡ࣮ࣜࢽࣥࢢࢶ࣮࡛ࣝ࠶ࡿࡇ࡜ࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽ㸪ࣔࣝ⃰ᗘࡢタᐃ࡛ࡁ࡞࠸⣲ᮦ࡬ࡢ ROS ࢔ࢵࢭ࢖ᒎ㛤ࡢྍ⬟ᛶࡀ♧ࡉࢀࡓ㸬ࡓࡔࡋΰྜ≀࡟ࡼࡗ࡚㸪᳜≀ᢳฟ≀ࡢࡼ࠺࡟㸪ࡑࡢෆᐜ≀ࡀ୙᫂░࡛࠶ࡿሙྜࡀ⪃࠼ࡽࢀ㸪ྠࡌྡ๓ࡢ⣲ᮦ࡟࠾࠸࡚ࡶࡑࡢᵓᡂᡂศࡸ⤌ᡂࡀ኱ࡁࡃ␗࡞ࡿྍ⬟ᛶࡀ࠶ࡿ㸬ࡇࡢࡼ࠺࡞ሙྜ࡟࠾࠸࡚ࡣ㸪ROS ࢔ࢵࢭ࢖࡛ᚓࡽࢀࡿ⤖ᯝ࡟ࡶ኱ࡁ࡞ᙳ㡪ࢆཬࡰࡍࡇ࡜ࡀ⪃࠼ࡽࢀࡿࡓࡵ㸪ㄗࡗࡓගᏳ඲ᛶ᝟ሗࢆᚓࡿࡇ࡜ࡀ↓࠸ࡼ࠺࡟⣽ᚰࡢὀពࡀᚲせ࡛࠶ࡿ㸬26
32

⾲ 5 ヨ㦂࡟⏝࠸ࡽࢀࡓ≀㉁ศ㢮ගẘᛶ≀㉁㸦40 ≀㉁㸧

໬⢝ရ㸦30 ≀㉁㸧

㸦17 ≀㉁㸧
4-Methyl-7-ethoxyco
umarin5-Methoxypsoralen
6-Methylcoumarin
7-Methoxycoumarin
8-Methoxypsoralen
Benzophenone
Bithionol
Dichlorophene
Fenticlorࡑࡢ௚≀㉁㸦16 ≀㉁㸧

㸦12 ≀㉁㸧
Acridine
Amiodarone HCl
Chlorpromazine HCl
Diclofenac Na
Enoxacin
Fenofibrateΰྜ≀㸦20 ≀㉁㸧

㸦11 ≀㉁㸧
Angelica root oil
Bergamot oil
Celery seed oil
Cumin seed oil
Grapefruit oil
Lemon oilLime oilOil parsley
St. John’s wort powder
Tagetes oil
Verbena oil⾲ 6㠀ගẘᛶ≀㉁㸦26 ≀㉁㸧
Hexachlorophene
Methyl ȕ-naphthyl
ketoneMethyl N-methyl
anthranilate
Muk ambretteMusk ketone
Musk xylene
p-Phenylenediamine
Triclocarban

Indomethacin
Ketoprofen
Piroxicam
Promethazine HCl
Sulfanilamide
Tetracycline HCl㸦13 ≀㉁㸧
1,3-Butylene glycol
2-Propanol
3-(-4-Methylbenzylidene) camphor
Ascorbic acid
Cetyl alcohol
Ethanol
Glycerine
Isopropyl myristate
Lauric acid
Propylene glycol
Sodium laurate
Sodium lauryl sulfate
Sulisobenzone
㸦4 ≀㉁㸧DMSO
Lactic acid
Methyl salicylate
Penicillin G㸦9 ≀㉁㸧
Carrot oil
Corn oilGinger oil
Lemongrass oil
Olive oilOrange oil
Rape seed oil
Safflower oil
Soybean oil

ROS ࢔ࢵࢭ࢖ヨ㦂⤖ᯝ (50 ȝg/mL)

ศ㢮

ឤᗘ

≉␗ᗘ

㝧ᛶ
ண ⋡

㝜ᛶ
ண ⋡

ṇ☜ᛶ

໬⢝ရ

100%
(17/17)76.9%(10/13)85.0%(17/20)100%(10/10)90.0%(27/30)

ࡑࡢ௚
≀㉁

100%
(12/12)

50.0%
(2/4)

85.7%
(12/14)

100%
(2/2)

87.5%
(14/16)ΰྜ≀

100%(11/11)

55.6%
(5/9)

73.3%
(11/15)

100%
(5/5)

80.0%
(16/20)50 ȝg/mL ࡛ヨ㦂ࢆᐇ᪋ࡋ㸪ุᐃᇶ‽ࡣᚑ᮶ἲ࡟ᚑࡗࡓሙྜࡢ⤖ᯝࢆ♧ࡍ㸬ឤᗘ㸸ගẘᛶ≀㉁ࡀ ROS ࢔ࢵࢭ࢖࡛㝧ᛶࢆ♧ࡍ๭ྜ㸦㸣㸧≉␗ᗘ㸸㠀ගẘᛶ≀㉁ࡀ ROS ࢔ࢵࢭ࢖࡛㝜ᛶࢆ♧ࡍ๭ྜ㸦㸣㸧㝧ᛶண ⋡㸸ROS ࢔ࢵࢭ࢖࡛㝧ᛶࡔࡗࡓ≀㉁ࡢ࠺ࡕ㸪ගẘᛶ≀㉁ࡢ๭ྜ㸦㸣㸧㝜ᛶண ⋡㸸ROS ࢔ࢵࢭ࢖࡛㝜ᛶࡔࡗࡓ≀㉁ࡢ࠺ࡕ㸪㠀ගẘᛶ≀㉁ࡢ๭ྜ㸦㸣㸧ṇ☜ᛶ㸸ROS ࢔ࢵࢭ࢖ࡢุᐃ⤖ᯝࡀගẘᛶ≀㉁㸪㠀ගẘᛶ≀㉁ࢆṇࡋࡃุᐃࡋࡓ๭ྜ㸦㸣㸧27
33

4.4 ග࢔ࣞࣝࢠ࣮ᛶண࡬ࡢ฼⏝໬⢝ရ⣲ᮦ࡟࠾࠸࡚㸪ගᏳ඲ᛶࡣගẘᛶ࡜ග࢔ࣞࣝࢠ࣮ᛶ࡟ศࡅࡽࢀࡿࡀ㸪ࡑࡢ࠺ࡕ㸪ග࢔ࣞࣝࢠ࣮ᛶࡣ㸪ගࢆ௓ࡋࡓ≀㉁࡜ࢱࣥࣃࢡ㉁ࡢ┤᥋⤖ྜࡲࡓࡣ㸪ග࡟ࡼࡿศゎ≀࡜ࢱࣥࣃࢡ㉁ࡢ⤖ྜ࡟ࡼࡿᢠཎᙧᡂࡀ㔜せ࡛࠶ࡿ㸬ග↷ᑕ࡟ࡼࡿບ㉳ගẘᛶ≀㉁⏘⏕᫬࡟Ⓨ⏕ࡍࡿ singlet oxygen ࡜ superoxide ࡣ㸪ග่⃭ᛶࢆច㉳ࡍࡿẘᛶບ㉳≀㉁ࡔࡅ࡛ࡣ࡞ࡃ㸪ග࢔ࣞࣝࢠ࣮ᛶࢆ♧ࡍ≀㉁࡛ࡶྠᵝ࡟Ⓨ⏕ࡋ㸪ග࢔ࣞࣝࢠ࣮ᛶ࡛ࡣ㸪ගࢱࣥࣃࢡ⤖ྜࡲࡓࡣග
ศゎࡢ࢚ࢿࣝࢠ࣮࡟ࡘ࡞ࡀࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ (Tokura, 2009)㸬୍⯡࡟㸪ග࢔ࣞࣝࢠ࣮ᛶࡢ
ᐃ⩏ࡀᐃࡲࡗ࡚࠾ࡽࡎ㸪⮫ᗋࡢ⌧ሙ࡟࠾࠸࡚ࡶගẘᛶ࡜ග࢔ࣞࣝࢠ࣮ᛶࡀ᫂☜࡟ศࡅࡽࢀ
࡚࠸࡞࠸ሙྜࡀከ࠸࡜⪃࠼ࡽࢀࡿ㸬ࡇࡢࡼ࠺࡞⌧≧࡟࠾࠸࡚ࡣ㸪ගẘᛶࢆ♧ࡉ࡞࠸ග࢔ࣞ
ࣝࢠ࣮ᛶ≀㉁ࡢ㑅ᐃࡀ㞴ࡋ࠸ࡓࡵ㸪ROS ࢔ࢵࢭ࢖ࡀస⏝ᶵᗎࡢほⅬ࠿ࡽග࢔ࣞࣝࢠ࣮ᛶ≀㉁ࡢ᳨ฟࡀྍ⬟࠿ྰ࠿ࢆ᳨ドࡍࡿࡇ࡜ࡣᐜ࡛᫆ࡣ࡞࠸㸬ࡑࡇ࡛ Onoue ࡽࡣ㸪ග࢔ࣞࣝࢠ࣮
ᛶ≀㉁࡜ࡋ࡚ሗ࿌ࡉࢀ࡚࠸ࡿ≀㉁ࡢ୰࡛㸪In vivo ගẘᛶヨ㦂࡟࠾࠸࡚㝜ᛶ⤖ᯝࡀሗ࿌ࡉࢀ
࡚࠸ࡿ≀㉁㸪ࡶࡋࡃࡣ 3T3 NRU-PT ࢆᐇ᪋ࡋ㸪㝜ᛶࡶࡋࡃࡣ␲㝧ᛶࢆ♧ࡋࡓ≀㉁ࢆග࢔ࣞࣝ
ࢠ࣮ᛶࡢᐤ୚ࡀ㧗࠸≀㉁࡜ࡋ࡚ศ㢮ࡋࡓ㸬ග࢔ࣞࣝࢠ࣮ᛶ≀㉁࡜ࡋ࡚ศ㢮ࡉࢀࡓ 18 ≀㉁ࢆ
⾲ 7 ࡟♧ࡋࡓ㸬ࡑࢀࡽ඲࡚ࡢ ROS ࢔ࢵࢭ࢖ࡢ⤖ᯝࡣ㝧ᛶ࡛࠶ࡗࡓ㸬ᚑࡗ࡚㸪ගẘᛶ࡟ග࢔ࣞࣝࢠ࣮ᛶࢆྵࡵࡓගᏳ඲ᛶࡢホ౯ࢶ࣮ࣝ࡜ࡋ࡚ ROS ࢔ࢵࢭ࢖ࢆ฼⏝ࡍࡿࡇ࡜ࡣጇᙜ࡜⪃࠼ࡽࢀࡿ(Onoue et al., perspnal communication)㸬
⾲ 7 ගẘᛶ㝜ᛶࡀሗ࿌ࡉࢀ࡚࠸ࡿග࢔ࣞࣝࢠ࣮ᛶ≀㉁ගẘᛶ㝜ᛶሗ࿌

In vivo assay (Guinea pigs)

In vitro assay㸦3T3 NRU-PT㸧

ග࢔ࣞࣝࢠ࣮ᛶ≀㉁࡜ࡋ࡚ሗ࿌
ࡉࢀ࡚࠾ࡾ㸪In
vivo and/or In vitroගẘᛶヨ㦂࡛㝜ᛶࢆ♧ࡍ໬ྜ≀㸦18 ≀㉁㸧

㝜ᛶ㸦10 ≀㉁㸧6-Methylcoumarin
Bithionol
Indomethacin
Ketprofen
Musk ambretteMusk xylene
Piroxicam
Sulfanilamide
Tribromosalicylanilide
Trichlorocarbanilide㝜ᛶ PIF < 2㸦5 ≀㉁㸧
Dichlorophene
Fenticlor
Hexachlorophene
Hydrochlorothiazide
Isoniazid

PIF: Photo irritation factor

28
34␲㝧ᛶ PIF > 2 and ൏ 5㸦3 ≀㉁㸧
Octyl dimethyl PABA
Omadine (Na salt)
Pyridoxine HCl

5 3Rs ࡬ࡢ㛵୚㸦ື≀⚟♴㠃࠿ࡽࡢጇᙜᛶ㸧
ROS ࢔ࢵࢭ࢖࡛ࡣ໬ྜ≀ࢆ཯ᛂᾮ࡟⁐ゎࡋ࡚ගࢆ↷ᑕࡋࡓ㝿࡟⏘⏕ࡉࢀࡿ ROS ࢆᐃࡍࡿヨ㦂⣔࡛࠶ࡾ㸪ື≀࠶ࡿ࠸ࡣື≀⏤᮶ࡢ⏕యᮦᩱࢆ౑⏝ࡋ࡞࠸ࡇ࡜࠿ࡽ㸪ROS ࢔ࢵࢭ࢖ࡢᐇ᪋࡟㝿ࡋ࡚ື≀⚟♴㠃ࡢၥ㢟ࡣᏑᅾࡋ࡞࠸㸬
ROS ࢔ࢵࢭ࢖ࡣ㸪2014 ᖺ 5 ᭶࡟බ㛤ࡉࢀࡓ་⸆ရࡢගᏳ඲ᛶ࢞࢖ࢻࣛ࢖ࣥ࡟࠾࠸࡚㸪ึᮇホ౯ࡢホ౯㡯┠ࡢࡦ࡜ࡘࡢ໬Ꮫⓗග཯ᛂᛶホ౯⣔࡜ࡋ࡚グ㍕ࡉࢀ㸪ROS ࢔ࢵࢭ࢖࡛㝜ᛶ࡜ุᐃࡉࢀࡓ㛤Ⓨ୰ࡢ་⸆ရ࡟㸪ࡉࡽ࡞ࡿගẘᛶヨ㦂ࢆ⾜࠺ᚲせ࡞࠸࡜ࡉࢀ࡚࠸ࡿ㸬ࡑࡢࡓࡵ㸪ROS ࢔ࢵࢭ࢖ࢆගᏳ඲ᛶホ౯࡟ᑟධࡍࡿࡇ࡜࡟ࡼࡾ㸪in vitro ගẘᛶヨ㦂࡛࠶ࡿ 3T3
NRU-PT ࡢヨ㦂ᩘࢆ๐ῶࡍࡿࡇ࡜ࡀ࡛ࡁ㸪ࡉࡽ࡟ in vivo ගẘᛶヨ㦂ࡢヨ㦂ᩘ๐ῶ࡟ࡘ࡞ࡀࡿ㸬

6 ヨ㦂᪉ἲࡢ᭷⏝ᛶ࡜ၥ㢟Ⅼ⣽⬊ࡸື≀ࢆ฼⏝ࡍࡿගᏳ඲ᛶホ౯⣔࡛ࡣ࢚ࢿࣝࢠ࣮ࡢ㧗࠸ UVB ࢆග※࡜ࡋ࡚౑⏝ࡍࡿࡇ࡜ࡀ㞴ࡋࡃ㸪ከࡃࡢሙྜࡣ UVA ࡢࡳࡢ↷ᑕࢆవ൤࡞ࡃࡉࢀࡿ㸬ࡇࡢࡓࡵ㸪UVB 㡿ᇦ࡟ࡋ࠿྾཰ࢆ᭷ࡉ࡞࠸໬ྜ≀࡟ᑐࡋ࡚ࡣഇ㝜ᛶホ౯ࢆ୚࠼ࡿࡇ࡜ࡀ࠶ࡿࡀ㸪ROS ࢔ࢵࢭ࢖ࡣࢩࣥࣉࣝ࡞≀ᛶホ౯⣔࡛࠶ࡿࡓࡵ࡟ UVB ࡢ↷ᑕࡶྍ⬟࡜࡞ࡿ㸬ࡲࡓ㸪⏕యヨᩱࢆᚲせ࡜ࡋ࡞࠸ࢩࢫࢸ࣒ࡢࡓࡵ㸪ࡑࡢ෌⌧ᛶࡶ㧗࠸㸬୍᪉㸪᪉ἲㄽⓗ࡞㝈⏺࡜ࡋ࡚ࡣ ROS ࢔ࢵࢭ࢖ࡣ໬ྜ≀ࢆ཯ᛂᾮ࡟⁐ゎࡋ࡚ගࢆ↷ᑕࡋࡓ㝿࡟⏘⏕ࡉࢀࡿ ROS 㔞ࢆ྾ගᗘࡢኚ໬࠿ࡽ
ᐃࡍࡿヨ㦂⣔࡛࠶ࡿࡓࡵ㸪཯ᛂᾮ୰࡛ᯒฟࡍࡿ⁐ゎᛶࡢప࠸≀㉁ࡸ⁐ゎ᫬࡟╔Ⰽࡀㄆࡵࡽ
ࢀࡿ໬Ꮫ≀㉁ࡣホ౯࡛ࡁ࡞࠸㸬ࡇࢀࡽㄢ㢟࡟ࡘ࠸࡚ࡢゎỴ⟇࡜ࡋ࡚ᨵⰋᆺ ROS ࢔ࢵࢭ࢖ࡸ
ヨ㦂ࢥࣥࢺ࣮ࣟࣝ⁐ᾮࡢタ⨨࡞࡝ࡀᥦ᱌ࡉࢀ࡚࠾ࡾ㸪ࡑࢀࡽ࡛ᚓࡽࢀࡓ▱ぢ࡟ࡘ࠸࡚ࡣ
ROS ࢔ࢵࢭ࢖ࡢ⤖ᯝุ᩿ࡢ᭦࡞ࡿຓࡅ࡟࡞ࡿ࡛࠶ࢁ࠺㸬ࡲࡓ㸪ROS ࢔ࢵࢭ࢖ࡣ௦ㅰ㐣⛬ࢆྵࡲ࡞࠸ࡇ࡜࠿ࡽ⿕㦂≀㉁ࡢ⏕యෆ௦ㅰ࡟౫ᏑࡍࡿගᏳ඲ᛶࣜࢫࢡࢆண

ࡍࡿࡇ࡜ࡀ㞴ࡋ࠸㸬ヨ㦂࡟⏝࠸ࡓ⃰ᗘ࡜㝧ᛶ/㝜ᛶุᐃ࡟ࡘ࠸࡚㸪㝧ᛶ⤖ᯝࡢุᐃ࡞ࡽࡧ࡟ 200 μM ⃰ᗘ࡛ࡢ㝜ᛶุᐃ࡟ࡣၥ㢟࡞࠸ࡀ㸪20 μM ⃰ᗘ࡛ࡢ㝜ᛶุᐃ࡟ࡣὀពࡍࡿᚲせࡀ࠶ࡿ㸬ࣂࣜࢹ࣮ࢩࣙࣥ
࣐ࢿࢪ࣓ࣥࢺࢳ࣮࣒ࡀ➨୕⪅ホ౯ጤဨ఍ࡢ່࿌ࢆཷࡅ࡚సᡂࡋࡓ᥎ዡࣉࣟࢺࢥ࣮࡛ࣝࡣᯒ
ฟ㸪╔Ⰽ࡟ࡼࡾ 200μM ุ࡛ᐃ࡛ࡁ࡞࠸ሙྜ࡟ 20 μM ࡢヨ㦂⤖ᯝࢆ᥇⏝࡛ࡁࡿ࡜ࡋ࡚࠸ࡿ㸬ࡋ࠿ࡋ࡞ࡀࡽ㸪⾜ᨻ࡬ࡢ⏦ㄳ᫬࡟ࡉࡽ࡞ࡿගẘᛶホ౯ࡣ୙せ࡜ุ᩿ࡍࡿ᰿ᣐ࡜ࡋ࡚ 20 μMࡢ㝜ᛶ⤖ᯝࢆ฼⏝ࡍࡿሙྜ࡟ࡣ㸪⿕㦂≀㉁ࡢග↷ᑕ᮲௳ୗ࡛ࡢᏳᐃᛶࢆ☜ㄆࡍࡿࡶࡢ࡜つᐃࡋ࡚࠸ࡿ㸬୍᪉㸪་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ㸦ཌ⏕ປാ┬㸪2014㸧࡛ࡣ ROS ࢔ࢵࢭ࢖࡟㛵ࡋ࡚㸪ࠕ200μM ࡢヨ㦂⃰ᗘ࡛㐺ษ࡞᮲௳ୗ࡛ᐇ᪋ࡉࢀࡓሙྜ㸪ࡇࡢヨ㦂ἲ࡛ࡢ㝜ᛶ⤖ᯝࡣගẘᛶࡢᠱᛕࡀ㠀ᖖ࡟ప࠸ࡇ࡜ࢆ♧ࡍ͐ࠖ࡜グ㍕ࡉࢀ㸪20 μM ࡛ࡢ㝜ᛶุᐃ⤖ᯝࡣࡉࡽ࡞ࡿගᏳ඲ᛶヨ㦂ࢆ୙せ࡜ࡍࡿ᰿ᣐ࡜ࡋ࡚᥇⏝ࡋ࡞࠸ࡇ࡜ࢆ♧ࡋ࡚࠸ࡿ㸬ࣂࣜࢹ࣮

29
35

ࢩࣙࣥሗ࿌᭩࡟ࡼࡿ࡜㸪ࣉࣞࣂࣜࢹ࣮ࢩ࡛ࣙࣥࡢ㝧ᛶ໬Ꮫ≀㉁ 11 ≀㉁ࡣ㸪200 μM ࡛඲≀㉁ࡀ㝧ᛶࢆ♧ࡋ㸪20 μM ࡛ࡣ 10 ≀㉁ࡀ㝧ᛶ⤖ᯝࢆ♧ࡋ㸪1 ≀㉁㸦8-MOP㸧ࡀ㝜ᛶࢆ♧ࡋࡓ㸬ࡲࡓ㸪ICH ࡢ S10 E W G ࡢᣦ᦬ࢆཷࡅ࡚㸬ࣂࣜࢹ࣮ࢩࣙࣥཧຍ᪋タ 1 ᪋タ࡟࡚ᐇ᪋ࡋࡓࣂ
ࣜࢹ࣮ࢩࣙࣥ࡟⏝࠸ࡓ 42 ≀㉁࡟ࡘ࠸࡚㸪20 μM ⃰ᗘ࡛ࡢ ROS ࢔ࢵࢭ࢖ࢆᐇ᪋ࡋࡓ⤖ᯝࡣ200 μM ⃰ᗘᐃ᫬࡟ẚ࡭ࡿ࡜཯ᛂࡣᑠࡉ࠿ࡗࡓࡀ㸪ࡍ࡭࡚ࡢ㝧ᛶ≀㉁࡛㝧ᛶࢆ♧ࡋ㸪8-MOPࡶྵࡵ㸪ഇ㝜ᛶ⤖ᯝࢆ࿊ࡋࡓ≀㉁ࡣ࡞࠿ࡗࡓ㸬200 μM ࡟ẚ࡭㸪20 μM ࡛཯ᛂࡀᑠࡉࡃ࡞ࡿ
⃰ᗘ࡟㛵㐃ࡋࡓせᅉ࡜ࡋ࡚㸪໬ྜ≀ࡢගᏳᐃᛶࡀ⪃࠼ࡽࢀࡓࡇ࡜㸪࡞ࡽࡧ࡟㸪20 μM ࡛ࡢഇ㝜ᛶ⤖ᯝࢆ♧ࡋࡓ 8-MOP ࡣ 200 μM ⃰ᗘ࡛ホ౯ྍ⬟࡛㝧ᛶ⤖ᯝࢆ♧ࡋࡓࡇ࡜࠿ࡽ㸪᥎ዡࣉࣟࢺࢥ࣮࡛ࣝࡣ 20μM ⃰ᗘࡢ⤖ᯝࡣ 200 μM ࡛ホ౯࡛ࡁ࡞࠸ሙྜ࡛㸪࠿ࡘ㸪 ᐃ᮲௳ୗ࡛⿕㦂≀㉁࡟ග࡟ࡼࡿศゎࡀㄆࡵࡽࢀ࡞࠸ሙྜ࡟㝈ᐃࡍࡿࡇ࡜࡟ࡼࡾ㸪ࣂࣜࢹ࣮ࢩࣙࣥࡢ⤖ᯝࡀṇ☜࡟཯ᫎࡉࢀ࡚࠸ࡿ㸬୍᪉㸪ICH S10 EWG ࡛ࡣ 20 μM ࡛ࡢ㝜ᛶ⤖ᯝࢆ་⸆ရ㛤ⓎရࡢගᏳ඲ᛶホ౯ࢆ୙せ࡜ࡍࡿุ᩿ࡢ᰿ᣐ࡜ࡋ࡚᥇⏝ࡍࡿࡇ࡜࡟ៅ㔜࡛࠶ࡾ㸪㝜ᛶ⤖ᯝࡣ 200
μM ࡛ࡢヨ㦂⤖ᯝࡢࡳࢆ᥇⏝ࡍࡿࡇ࡜࡜࡞ࡗࡓ㸬௒ᚋ㸪20 μM ⃰ᗘ࡛ࡢヨ㦂⤖ᯝࡢ㞟✚࠶ࡿ
࠸ࡣ 200 μM ⃰ᗘ࡛ࡢྍ⁐໬➼ࡢᯒฟ㜵Ṇᢏ⾡ࡢᛂ⏝࡟ࡼࡾ㸪ヨ㦂⤖ᯝࡢ฼⏝᪉ἲࡀ」ᩘ࠶
ࡿࡇ࡜࡟ࡼࡿΰ஘ࡀゎᾘࡉࢀࡿࡇ࡜ࡀᮃࡲࢀࡿ㸬ΰྜ≀࠾ࡼࡧศᏊ㔞୙᫂ヨᩱ࡟ࡘ࠸࡚ࡣ㸪μg/mL ༢఩࡛ࡢ ROS ࢔ࢵࢭ࢖࡟㛵ࡍࡿሗ࿌ࡀ
࡞ࡉࢀ࡚࠸ࡿ㸬་⸆ရ࡛ࡣ ROS ࢔ࢵࢭ࢖ࡣගᏳ඲ᛶࡢึᮇホ౯࡜ࡋ࡚㸪ගᏳ඲ᛶホ౯ࡢせྰุ᩿࡟฼⏝ྍ⬟࡛࠶ࡾ㸪ICH ຍ┕ᅜࡢつไᙜᒁ࡟ࡼࡾཷࡅධࢀࡽࢀࡿ㸬໬Ꮫ≀㉁࠾ࡼࡧ㎰⸆࡛ࡣ㸪ROS ࢔ࢵࢭ࢖ࡣග཯ᛂᛶࢆホ౯ࡍࡿࡇ࡜࡛㸪₯ᅾⓗ࡞ගẘᛶࡢࣁࢨ࣮ࢻ᳨ฟࡢᡭἲ࡜ࡋ࡚᭷⏝࡛࠶ࢁ࠺㸬໬⢝ရ࡛ࡣ㸪ROS ࢔ࢵࢭ࢖࠾ࡼࡧᨵⰋᆺ ROS ࢔ࢵࢭ࢖ࢆ㐺⏝ࡍࡿࡇ࡜࡛㸪ගẘᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶ≀㉁ࡢ᳨ฟࡀྍ⬟࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿ㸦Onoue et al., 2013㸧㸬ࡋ࠿ࡋ࡞ࡀ
ࡽ㸪ග໬Ꮫⓗ཯ᛂᛶヨ㦂࡛࠶ࡿ ROS ࢔ࢵࢭ࢖ࡢ≉ᛶ࠿ࡽ㸪㐺ษ࡟ᐇ᪋ࡉࢀࡓ ROS ࢔ࢵࢭ࢖࡛㝜ᛶ࡛࠶ࢀࡤගẘᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶࢆ♧ࡉ࡞࠸࡜ุ᩿ࡋ࡚ᕪᨭ࠼࡞࠸ࡀ㸪ROS ࢔ࢵࢭ࢖࡟࠾ࡅࡿ㝧ᛶࡣග཯ᛂᛶࢆ᭷ࡍࡿࡇ࡜ࢆ♧ࡍࡶࡢ࡛࠶ࡗ࡚㸬ගẘᛶ㸪ග࢔ࣞࣝࢠ࣮ᛶࡢ
᭷↓ࡢホ౯ࡣู㏵ホ౯ࡉࢀࡿ࡭ࡁ࡛࠶ࡿ㸬

7 ⤖ㄽ
ROS ࢔ࢵࢭ࢖ࡣ໬Ꮫ≀㉁࡟ᨃఝኴ㝧ගࢆ↷ᑕࡋ㸪singlet oxygen ࡜ super oxide anion ࡢ⏘⏕ࡢ᭷↓ࢆㄪ࡭ࡿග཯ᛂᛶヨ㦂ࡢ୍ࡘ࡛࠶ࡿ㸬7 ᪋タ࡟ࡼࡾᐇ᪋ࡉࢀࡓከ᪋タࣂࣜࢹ࣮ࢩࣙ
ࣥヨ㦂࡟࠾࠸࡚㸪ROS ࢔ࢵࢭ࢖ࡣ㸪᪋タෆࡢ᪥ෆᕪ/᪥㛫ᕪ࠾ࡼࡧ᪋タ㛫ᕪࡀᑠࡉࡃ㸪ỗ⏝ᛶ࡟ඃࢀ࡚࠸ࡿヨ㦂ἲ࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓ㸬ࡲࡓ㸪㞴⁐ᛶࡢࡓࡵホ౯࡛ࡁ࡞࠿ࡗࡓ⿕㦂≀㉁ࢆ㝖ࡃ࡜㸪ROS ࢔ࢵࢭ࢖࡟ࡼࡿගẘᛶ≀㉁ࡢ㝧ᛶ᳨ฟ⋡ࡣ 100%࡛࠶ࡾ㸪ഇ㝜ᛶ⤖ᯝࡀ࡞࠸ࡇ࡜࠿ࡽ㸪ROS ࢔ࢵࢭ࢖ࡣගẘᛶ࣏ࢸࣥࢩࣕࣝࡢホ౯࡟᭷⏝࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓ㸬30
36ࡉࡽ࡟ ROS ࢔ࢵࢭ࢖ከ᪋タࣂࣜࢹ࣮ࢩࣙࣥࡢ➨୕⪅ホ౯ጤဨ఍ࡣࡑࡢሗ࿌᭩㸦Spielmann
et al., 2013㸧࡟࠾࠸࡚㸪ࠕጤဨ఍ࡣ㸪ROS ࢔ࢵࢭ࢖ࡢ෌⌧ᛶ࠾ࡼࡧண

ᛶࡀ୍㐃ࡢගᏳ඲ᛶ

ヨ㦂ᐇ᪋࡞ࡽࡧ࡟་⸆ရࡢ◊✲㛤Ⓨ࡟࠾ࡅࡿពᛮỴᐃᡓ␎࡛ ROS ࢔ࢵࢭ࢖ࡀ฼⏝ࡉࢀࡿࡇ࡜ࢆᨭᣢࡍࡿࡇ࡜࡟༑ศ࡛࠶ࡿ࡜⤖ㄽࡍࡿ㸬ࡇࡢᡓ␎࡟࠾࠸࡚㸪ROS ࢔ࢵࢭ࢖࡛㝧ᛶ㸪ᙅ࠸㝧ᛶࢆ♧ࡍ࠿㸪ุᐃ୙⬟ࡢሙྜ࡟ࡣ㸪3T3 NRU-PT (OECD Test Guideline 432)࡞࡝ࡢḟࡢࣞ࣋ࣝࡢ in vitro ヨ㦂⣔࡟㐍ࡵ㸪㝜ᛶࡢሙྜ࡟ࡣື≀࡛ࡢヨ㦂ࡸ௚ࡢヨ㦂ࡣせồࡉࢀ࡞࠸㸬ጤဨ఍ࡣࡲࡓ㸪ROS ࢔ࢵࢭ࢖ࡣගᏳ඲ᛶホ౯࡟せࡍࡿᮇ㛫㸪㈝⏝࠾ࡼࡧ౑⏝ື≀ᩘࡢ๐ῶ࡟ࡘ࡞ࡀࡿ࡜⤖ㄽࡋࡓ㸬ࡉࡽ࡟ ROS ࢔ࢵࢭ࢖ࢆගᏳ඲ᛶホ౯ᡓ␎࡟⤌ࡳධࢀࡿࡇ࡜ࡣ 3T3
NRU-PT ࡸࡑࢀ࡟⥆ࡃື≀࡛ࡢヨ㦂ࡀᚲせ࡜࡞ࡿ໬ྜ≀ᩘࢆ᭷ព࡟ῶᑡࡉࡏࡿࡇ࡜࡟ࡘ࡞
ࡀࡿ㸬ࠖ࡜⤖ㄽࡋࡓ㸬ᮏࣂࣜࢹ࣮ࢩࣙࣥ⤖ᯝ࡟ᇶ࡙ࡁ㸪ICH ࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ࡟࠾࠸࡚㸪ROS ࢔ࢵ
ࢭ࢖ࡣ඲㌟࠾ࡼࡧ⓶⭵㐺⏝⸆࡟࠾ࡅࡿගẘᛶホ౯ࡢせྰุ᩿࡟฼⏝ྍ⬟࡞ヨ㦂ἲࡢ୍ࡘ࡜
ࡋ࡚᥇ᢥࡉࢀࡓ㸦Step 4㸪2013 ᖺ 11 ᭶㸧㸬௒ᚋ㸪ROS ࢔ࢵࢭ࢖࡛㝜ᛶࡢሙྜࡣ㸪ࡉࡽ࡞ࡿගẘᛶホ౯ࡀ୙せ࡜࡞ࡾ㸪ගẘᛶヨ㦂ᩘ࡜ࡑࡢ㈝⏝ࡢ๐ῶຠᯝࡀᮇᚅࡉࢀࡿ㸬ࡉࡽ࡟᭱㏆ࡢ᳨ウ࡟࠾࠸࡚㸪ග࢔ࣞࣝࢠ࣮ᛶ≀㉁ࡶ ROS ࢔ࢵࢭ࢖᳨࡛ฟࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜ࡢ▱ぢࡀᚓࡽࢀ㸪ROS ࢔ࢵࢭ࢖ࡣගẘᛶࡔࡅ࡛࡞ࡃ㸪ග࢔ࣞࣝࢠ࣮ᛶࡶྵࢇࡔගᏳ඲ᛶࡢホ౯ࢶ࣮ࣝ࡜ࡋ࡚฼⏝ࡉࢀࡿࡇ࡜ࡀᮇᚅࡉࢀࡿ㸬໬⢝ရཎᩱ࡟ࡘ࠸࡚ΰྜ≀࠾ࡼࡧศᏊ㔞୙᫂ヨᩱ࡬ࡢ㐺⏝ྍ⬟ᛶࡀ♧ࡉࢀ㸪໬⢝ရཎᩱ㸪୍⯡໬Ꮫ≀㉁࡞ࡽࡧ࡟㎰⸆࡟ࡘ࠸࡚
ROS ࢔ࢵࢭ࢖ࡀ₯ᅾⓗ࡞ගᏳ඲ᛶ࡟㛵ࡍࡿࣁࢨ࣮ࢻ᳨ฟࡢᡭἲ࡜ࡋ࡚᭷⏝࡛࠶ࡿࡇ࡜ࡀᮇᚅࡉࢀࡿ㸬

8 ⏝ㄒࡢゎㄝ
Chromophore㸦ࢡࣟࣔࣇ࢛࢔㸧㸸ྍどග࠶ࡿ࠸ࡣ UV ࢆ྾཰ࡍࡿศᏊࡢ㒊ศᵓ㐀㸬
Internal conversion㸦ෆ㒊㌿⛣㸧㸸࠶ࡿศᏊࡀࡼࡾ࢚ࢿࣝࢠ࣮ࡢ㧗࠸㸪ࡶࡋࡃࡣప࠸᣺ືບ㉳≧ែ࡟㑄⛣ࡍࡿࡇ࡜㸬Intersystem crossing㸦㡯㛫஺ᕪ㸧㸸㧗࠸᣺ືບ㉳≧ែࡢศᏊ࠿ࡽࡢࢫࣆࣥኚ᥮࡟కࡗ࡚㏆ࡃ࡟ᏑᅾࡍࡿศᏊࡀ␗࡞ࡿ᣺ືບ㉳≧ែ࡟㑄⛣ࡍࡿࡇ࡜㸬

9 ཧ⪃ᩥ⊩
Andersen KE, Lindskov R (1984) Recall of UVB-induced erythema in breast cancerpatient receiving multiple drug
chemotherapy. Photodermatol, 1, 129-132.
Bauer D, Averett LA, De Smedt A, Kleinman MH, Muster W, Pettersen BA, Robles C (2014), Standardized UV-vis
spectra as the foundation for a threshold-based, integrated photosafety evaluation. Regul Toxicol Pharmacol, 68(1),70-75.

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Boccumini LE, Fowler CL, Campbell TA, Puertolas LF, Kaidbey KH (2000) Photoreaction potential of orally
administered levofloxacin in healthy subjects. Ann Pharmacother, 34(4), 453-458.
Dam C, Bygum A (2008) Subacute cutaneous lupus erythematosus induced or exacerbated by proton pump inhibitors.
Acta Derm Venereol, 88(1), 87-89.
Dillaha CJ, Jansen GT, Honeycutt WM, Bradford AC (1983) Selective cytotoxic effect of topical 5-fluorouracil. Arch
Dermatol, 119(9), 774-783.
Henry B, Fotib C, Alsanteb K (2009) Can light absorption and photostability data be used to assess the photosafety
risks in patients for a new drug molecule? J Photochem Photobiol B, 96(1), 57-62.
Kirkup ME, Narayan S, Kennedy CTC (2003) Cutaneous recall reactions with systemic fluorouracil. Dermatology,
206(2), 175-176.
Kleinman MH, Smith MD, Kurali E, Kleinpeter S, Jiang K, Zhang Y, Kennedy-Gabb SA, Lynch AM, Geddes CD(2010) An evaluation of chemical photoreactivity and the relationship to phototoxicity. Regul Toxicol Pharmacol,
58(2), 224-232.
Ljunggren B, Sjövall P (1986) Systemic quinine photosensitivity. Arch Dermatol, 122(8), 909-911.
Miolo G, Marzano C, Gandin V, Palozzo AC, Dalzoppo D, Salvador A, Caffieri S (2011) Photoreactivity of
5-Fluorouracil under UVB Light. Chem Res Toxicol, 24(8), 1319–1326.
Nishida H, Hirota H, Seto Y, Suzuki G, Kato M, Kitagaki M, Sugiyama M, Kouzuki H, Onoue S (2015) Non-animal
photosafety screening for complex cosmetic ingredients with photochemical and photobiochemical assessment tools.
Regul Toxicol Pharmacol, 72(3):578-585.
Onoue S, Tsuda Y (2006) Analytical studies on the prediction of photosensitive/phototoxic potential ofpharmaceutical substances. Pharm Res 23(1), 156-164Onoue S, Igarashi N, Yamada S, Tsuda Y (2008a)
High-throughput reactive oxygen species (ROS) assay: an enabling technology for screening the phototoxic potentialof pharmaceutical substances. J Pharm Biomed Anal, 46(1), 187-193.
Onoue S, Yamauchi Y, Kojima T, Igarashi N, Tsuda Y (2008b) Analytical studies on photochemical behavior ofphototoxic substances; Effect of detergent additives on singlet oxygen generation. Pharm Res, 25(4), 861–868.
Onoue S, Seto Y, Gandy G, Yamada, S (2009) Drug-Induced Phototoxicity; An Early In Vitro Identification of
Phototoxic Potential of New Drug Entities in Drug Discovery and Development. Curr Drug Saf, 4(2), 123-136.
Onoue S, Ochi M, Gandy G, Seto Y, Igarashi N, Yamauchi Y, Yamada S (2010) High-throughput screening system
for identifying phototoxic potential of drug candidates based on derivatives of reactive oxygen metabolites. Pharm
Res, 27(8), 1610-1619.
Onoue S, Hosoi K, Wakuri S, Iwase Y, Yamamoto T, Matsuoka N, Nakamura K Toda T, Takagi H, Osaki N,
MatsumotoY, Kawakami S, Seto Y, Kato M, Yamada S, Ohno Y and Kojima H (2013a) Establishment andintra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety
evaluation. J Appl Toxicol, 33(11), 1241–1250.
Onoue S, Suzuki G, Kato M, Hirota M, Nishida H, Kitagaki M, Kouzuki H, Yamada S (2013b) Non-animal
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Nakamuraj K, Ohno Y, Kojima H (2014a) Intra-/inter-laboratory validation study on reactive oxygen species assay
for chemical photosafety evaluation using two different solar simulators, Toxicol In Vitro, 28(4), 515–523
Onoue S, Kato M, Yamada S (2014b) Development of an albuminous reactive oxygen species assay for photosafety
evaluation under experimental biomimetic conditions, J Appl Toxicol, 34(2), 158–165
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http://www.jacvam.jp/files/news/ROS_Assay_full%2020130920%20atlas_fourth%20data.pdf (accessed May,
2015)ROS assay validation management team (2013b) Validation report for the international validation study on
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evaluation of poorly water-soluble chemicals. Toxicol In Vitro, 27(6), 1838–1846
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WW, Maurer T, Moldenhauer F, Moore L, Pape WJ, Pfanenbecker U, Potthast J, De Silva O, Steiling W, Willshaw A.
(1994) EEC/COLIPA project on in vitro phototoxicity testing: First results obtained with a Balb/c 3T3 cell
phototoxicity assay. Toxicol In Vitro., 8(4), 793-796.
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Gerberick F, Liebsch M, Lovell WW, Maurer T, Pfannenbecker U, Potthast JM, Csato M, Sladowski D, Steiling W,
Brantom P. (1998a) The International EU/COLIPA In Vitro Phototoxicity Validation Study: Results of Phase II(Blind Trial). Part 1: The 3T3 NRU Phototoxicity Test. Toxicol In Vitro, 12(3), 305-327.
Spielmann H, Stokes WS, Horii I, Kim BH (2013) Peer Review Panel Evaluation of the Reactive Oxygen Species(ROS) Photosafety Assay, http://www.jacvam.jp/files/doc/02_03/02_03_C1.pdf (accessed May, 2015)
Tokura Y (2009) Photoallergy. Expert Rev Dermatol, 4(3), 263-270.

ᑿୖㄔⰋ㸪୰ᮧ࿴ᕷ㸦2011㸧 ගẘᛶ㸪♫ᅋἲே᪥ᮏ⸆⌮Ꮫ఍⦅㞟ࠕᐇ㦂⸆⌮Ꮫ ๰⸆◊✲ࡢࢫࢺࣛࢸࢪ࣮
ୗࠖ㸦㔠ⰾᇽ㸧ཌ⏕ປാ┬㸦2010㸧 㔜⠜๪స⏝⑌ᝈูᑐᛂ࣐ࢽࣗ࢔ࣝ ᡭ㊊⑕ೃ⩌㸦http://www.info.pmda.go.jp/juutoku/file/jfm1003014.pdf㸪2015 ᖺ 2 ᭶࢔ࢡࢭࢫ☜ㄆ㸧ཌ⏕ປാ┬㸦2014㸧 ་⸆ရࡢගᏳ඲ᛶホ౯࢞࢖ࢻࣛ࢖ࣥ࡟ࡘ࠸࡚㸦⸆㣗ᑂᰝⓎ 0521 ➨ 1 ྕ㸪ᖹᡂ 26 ᖺ 5
᭶ 21 ᪥㸧

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10 Appendix
Appendix 1 Executive Summary of “Peer Review Panel
Evaluation of the Reactive Oxygen Species (ROS) Photosafety
Assay” (16 October 2013)
Executive Summary
The Japanese Center for the Validation of Alternative Methods (JaCVAM) convened an
independent scientific peer review panel to evaluate the validation status of the Reactive
Oxygen Species (ROS) assay in accordance with established international criteria (OECD,2005). The ROS assay is a test method proposed as a component of an integrated
photosafety testing strategy to evaluate whether test substances such as pharmaceuticals
have the potential to cause phototoxicity.
The panel met initially in February and again in August 2013 in Tokyo, Japan. The panel
considered the reports of two international validation studies and a proposed ROS ssay
protocol at their initial meeting. The panel subsequently reviewed updated versions of the
ROS Assay protocol and the validation study reports as revised by the Validation
Management Team (VMT). This report summarizes the panel’s final evaluation andconclusions.
Overall conclusion: The panel concluded that the reproducibility and predictivity of the
ROS assay is sufficient to support its use in an integrated photosafety testing and decision
strategy for drug research and development. In this strategy, negative results in the ROS
assay would not require further testing in animals or other tests, while positive, weakly
positive, and inconclusive results would proceed to the next level of testing in an in vitro test
system such as the 3T3 Phototoxicity Assay (OECD Test Guideline 432). The panel also
concluded that use of the ROS assay could potentially provide significant savings in time,
cost and reduced animal use for photosafety assessments. Furthermore, incorporating the
ROS assay into a photosafety testing strategy is expected to significantly reduce the overall
number of substances that would
require additional testing in the in vitro 3T3 Phototoxicity Assay and subsequent testing inanimals.
Regulatory rationale: The panel concluded that the ROS Assay is applicable for use within
the ICH regulatory testing framework for photosafety evaluation of pharmaceutical products.
Regulatory authorities (e.g. PMDA/MHLW, U.S. FDA, EMEA) require non-clinicalphotosafety testing prior to approving First-in-Human Phase I studies so that appropriate
precautions and observations can be taken during initial human studies. Such non-clinicalphotosafety testing typically includes an assessment of the potential for a drug to cause
phototoxic reactions, which are characterized clinically by dermal redness, swelling, irritation,and inflammation. The panel
also agreed that the ROS assay is applicable to in-house drug research and development.
Scientific rationale: The panel recognized that ROS production is the most importantmechanism for chemically-induced phototoxicity, and is therefore a critical pathway initiating
event leading to phototoxicity. The ROS assay quantitatively measures two common
reactive oxygen species generated by photoreactive chemicals after exposure to simulated
sunlight. In this validation study, chemicals that did not produce sufficient ROS to meet thephotoreactivity threshold classification criteria for the ROS assay are uniformly
non-phototoxic, while chemicals that met or exceeded the photoreactivity classification
criteria include all known phototoxicants. Therefore if a chemical is not photoreactive in the

34
40

ROS assay, it is unlikely that phototoxicity will occur in living systems.
Limitations: The panel noted that the ROS assay assesses chemical photoreactivity in a
nonbiological system, and therefore may overpredict phototoxicity potential since it does notassess the direct interaction of chemicals with biological tissues. The assay may also
overestimate the skin for phototoxic reactions to occur, or photodegradation may occur.
Accordingly, positive results in the ROS assay are generally recommended for further
evaluation in a photosafety testing strategy.
Validation study reference chemicals: The panel agreed that the reference chemicals
selected for the validation studies were appropriate and sufficiently representative of thechemicals likely to be evaluated in the assay. The 42 reference chemicals incorporated most
known human phototoxicants and included 23 known positives and 19 negatives. The
chemicals were backed by data from human patch testing and in vitro 3T3 phototoxicity
assay results. All data from the validation studies were made available in the validation
study reports.
Assay Reproducibility: The panel concluded that the assay had excellent reproducibility
both within and between laboratories for the 42 reference chemicals evaluated in thevalidation studies. Additionally, the positive and negative control chemicals had 100%
reproducibility within and between laboratories based on classification outcome, whichfurther supports the reproducibility of the ROS assay.
Test method predictivity: After reviewing analyses provided in the validation study reports,
the panel agreed that conducting a single assay per chemical provided optimal predictivity.
The panel concluded that the classification criteria for test outcomes have been
appropriately optimized to avoid false negatives while minimizing false positives. The panel
also noted that chemicals positive for both reactive oxygen species were uniformly
phototoxic.
Data quality: The panel agreed that the high level of within and between laboratory
reproducibility suggested a consistently high level of quality of the validation studies. While
the studies were not conducted in strict accordance with GLPs, most of the labs were GLP
certified. The validation management team also confirmed that quality control audits foundthat validation report data accurately reflected the raw data results.
Test method protocols: The panel considered the test method protocols used for the twovalidation studies and key aspects of a proposed standardized ROS assay protocol. The
panel recommended that the solar simulator should be equipped with an appropriate
temperature control unit or fan since ROS production can be influenced by temperature. The
panel concluded that the list of proficiency chemicals provided in the test method protocol
for laboratories to use to demonstrate ability to perform the assay was appropriate. The
panel recommended that each lab should develop historical positive and negative control
value acceptance ranges that can be used to determine the acceptability of an individual
test. The panel also agreed with the appropriateness of the reference chemicals identified
for qualification of solar simulators other than the two used in the validation studies.
Applicability domain: The applicability domain of the ROS assay is currently restricted toonly those chemicals that meet the solubility criteria outlined in the protocol. The panel
recommended that as experience is gained from use of the ROS assay, the applicability
domain could be more fully described in terms of physicochemical properties and/or
chemical classes. This will contribute to increased efficiency by providing criteria that can beused to identify whether a chemical may be satisfactorily tested in the ROS assay, or
whether an alternate assay should be used initially.

35
41

Appendix 2 List of reasons for chemical selection
NO.

CompoundReasons for chemical selection
5-FU was reportedly a phototoxic chemical in humans [Dillaha et al., 1983], but it
was negative in the 3T3NRU-PT [Kleinman et al., 2010 and Onoue et al., 2010].
Reported causes of human phototoxicity in 5-FU include photocytotoxicity induced
by UV-B alone [Kirkup M.E. et al., 2003 and Andersen K.E. et al., 1984] and/or
ROS generation derived from UV-B induced photodegradation [Miolo G. et al.,2011]. 5-FU absorbs mainly UV-B (290–320 nm) within the range of natural
sunlight (Appendix 7), UV-B irradiation might be essential for photochemical
activation of 5-FU.

Therefore it was unknown whether 5-FU was a phototoxic

chemical, and high quality human data was not available. 5-FU was selected inorder to provide information on the limits of the ROS assay. Photosensitivity was
I-1

5-Fluorouracil (5-FU)

mentioned on both the US and the JPN label.
8-MOP was selected from the list of phototoxic chemicals used in the 3T3NRU-PT
validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

I-2

8-Methoxy psoralen (8-MOP)

Photosensitivity was mentioned on both the US and the JPN label.
Amiodarone HCl was selected from the list of phototoxic chemicals used in the
3T3NRU-PT validation study. Human data for this chemical was described as
positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,

I-3

Amiodarone HCl

1994a].

Photosensitivity was mentioned on both the US and the JPN label.

Chlorpromazine HCl was selected from the list of phototoxic chemicals used in the
3T3NRU-PT validation study. Human data for this chemical was described as
positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,I-4

Chlorpromazine HCl

1994a].

Photosensitivity was mentioned on both the US and the JPN label.

Diclofenac was selected as a phototoxic chemical for human per the article byPrzybilla et al [1987].
I-5

Diclofenac

Photosensitivity was mentioned on both the US and the

JPN label.
Doxycycline HCl was selected from the list of phototoxic chemicals used in the
3T3NRU-PT validation study. Human data for this chemical was described as
positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,

I-6

Doxycycline HCl

1994a].

Photosensitivity was mentioned on both the US and the JPN label.

Furosemide was selected from the list of phototoxic chemicals used in the
I-7

Furosemide

3T3NRU-PT validation study. Human data for this chemical was described as3642

positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,
1998a].

Although the 2011 US label did mention photosensitivity, 2012 US label

did not. Photosensitivity was mentioned on the JPN label.
Ketoprofen was selected from the list of phototoxic chemicals used in the
3T3NRU-PT validation study. Human data for this chemical was described as
positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,I-8

Ketoprofen

1998a].

Photosensitivity was mentioned on both the US and the JPN label.

Levofloxacin was selected as a phototoxicity positive chemical for human per thearticle by Boccumini et al [2000].
I-9

Levofloxacin

Photosensitivity was mentioned on both the US

and the JPN label.
Norfloxacin was selected from the list of phototoxic chemicals used in the
3T3NRU-PT validation study. Human data for this chemical was described as
positive in the article on the 3T3 NRU-PT validation study [Spielmann et al,

I-10

Norfloxacin

1998a].

Photosensitivity was mentioned on both the US and the JPN label.

Omeprazole was selected as a phototoxic chemical for human per to the article by
Dam et al [2008].
I-11

Omeprazole

Photosensitivity was mentioned on both the US and the JPN

label.
Quinine HCl was used as positive control according to the method of Onoue et al.
[2008a].

Quinine HCl was classified as a phototoxic chemical for human per thearticle by Ljunggren et al [1986].
I-12

Quinine HCl

There was a mention of the photosensitivity in

the US label.
Sulizobenzone was used as negative control according to the method of Onoue etal. [2008a].

Sulisobenzone was classified as a non-phototoxic chemical, because

Human data for this chemical was described as negative in the article on the 3T3
I-13

Sulisobenzone

NRU-PT validation study [Spielmann et al, 1998b].

37
43

Appendix 3 Test chemicals for the Phase 1 study
UV/vis absorption b)

No.

Chemical name

CAS No.a)

MEC

Ȝmax

(L/mol/cm)(nm)

in vivo

ROS

3T3

assay

NRU

Animal

Human

I-1

5-FU

51-21-81800 c)

290 d)- 1)- 2, 3)

NA

+? 4)

I-2

8-MOP

298-81-7

3631

300

+ 1)

+ 5)

+ 5)

+ 5)

I-3

Amiodarone HCl

19774-82-4

5400

290 d)

+ 3)

+ 5)

+ 5)

+ 5)

I-4

Chlorpromazine HCl

69-09-0

1746

304

+ 1)

+ 5)

+ 5)

+ 5)

I-5

Diclofenac

15307-79-6

7800 c)

290 d)

+ 1)

+ 3)

+ 6)

+ 7)

I-6

Doxycycline HCl

10592-13-93715

290 d)

+ 1)

+ 5)

+ 5)

+ 5)

I-7

Furosemide

54-31-92650

290 d)+ 1)+/- 3,8,9)

NA

+ 8)

I-8

Ketoprofen

22071-15-4

2092

290 d)

+ 1)

+ 8)

- 8)

+ 8)

I-9

Levofloxacin

100986-85-4

13000 c)

333

+ 10)

+ 10)

+ 11)

+ 12)

I-10

Norfloxacin70458-96-7

3562

323

+ 1)

+ 3)

+ 8)

+ 8)

I-11

Omeprazole

73590-58-6

15000 c)

301

+ 1)

+/- 3)

NA

+ 13)

I-12

Quinine HCl

6119-47-7

1938

330

+ 1)

+ 3)

+ 6)

+ 14)

I-13

Sulisobenzone

4065-45-6

3519

290 d)

- 1)

- 3)

NA

- 15)

5-FU: 5-fluorouracil, 8-MOP䠖8-methoxy psoralen+ : Positive, - : Negative, +/- : Equivocal, NA : Not available, ? : uncleara) CAS No.: Chemical abstracts service registry number, b) The UV/vis absorbance (290-700 nm) of chemicals wasmeasured in 20 mM phosphate buffer (pH 7.4). Test chemicals were dissolved in dimethylsulfoxide (DMSO) at 10mM and diluted with 20 mM phosphate buffer (pH 7.4). Final concentration of DMSO was unified to 0.5%. c) Molar
extinction coefficient (MEC) of 5-FU, diclofenac, levofloxacin, and omeprazole were extracted from the articles ofOnoue et al. (2008a) and Seto et al. (2011).
d) Ȝmax (nm) was a wavelength at which the UV/vis absorbance shows a peak between 290 and 700 nm. Ȝmax (nm)
was indicated as 290 nm in the case where the peak absorption is located below 290 nm and the maximum absorption
is at 290 nm.
1) Onoue et al., 2008a, 2) Kleinman et al., 2010, 3) Onoue et al., 2010, 4) Dillaha et al., 1983, 5) Spielmann et al.,1994a, 6) Spielmann et al., 1994b, 7) Przybilla et al., 1987, 8) Spielmann et al., 1998a, 9) Peters et al., 2002, 10) Setoet al., 2011, 11) Wagai et al., 1992, 12) Boccumini et al., 2000, 13) Dam et al., 2008, 14) Ljunggren et al., 1986, 15)
Spielmann et al., 1998b38
44

Appendix 4 List of reasons for chemical selection
NO.

Chemical nameReasons for chemical selection

Phototoxic drugs
Acridine was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
Acridine (No. II-1) and acridine HCl (No. II-2) were tested both as a free form and a salt
in order to test whether the aqueous solubility of chemicals might limit the predictive
II-1

Acridine

power of the ROS assay.
Acridine HCl was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
Acridine (No. II-1) and acridine HCl (No. II-2) were tested both as a free form and a salt
in order to test whether the aqueous solubility of chemicals might limit the predictive

II-2

Acridine HCl

power of the ROS assay.
Amiodarone HCl was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-3

Amiodarone HCl

Photosensitivity

was mentioned on both the US and the JPN label.
Chlorpromazine HCl was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-4

Chlorpromazine HCl

Photosensitivity

was mentioned on both the US and the JPN label.
Doxycycline HCl was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-5

Doxycycline HCl

Photosensitivity

was mentioned on both the US and the JPN label.
Fenofibrate was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].

Although the 2011 USlabel did mention photosensitivity, 2012 US label did not. Photosensitivity was
II-6

Fenofibrate

mentioned on the JPN label.

II-7

Furosemide

Furosemide was selected from the list of positive chemicals used in the 3T3 NRU-PT

39
45

validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].

Although the 2011 USlabel did mention photosensitivity, 2012 US label did not. Photosensitivity wasmentioned on the JPN label.
Ketoprofen was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
II-8

Ketoprofen

Photosensitivity wasmentioned on both the US and the JPN label.
6-Methylcoumarine was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in the

II-9

6-Methylcoumarine

article on the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
8-MOP was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-10

8-MOP

Photosensitivity wasmentioned on both the US and the JPN label.
Nalidixic acid was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
Nalidixic acid (II-11) and nalidixic acid (Na salt) (II-12) were tested both as a free form
and a salt in order to test whether the aqueous solubility of chemicals might limit thepredictive power of the ROS assay. Photosensitivity was mentioned on both the US and

II-11

Nalidixic acid

the JPN label.
Nalidixic acid (Na salt) was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
Nalidixic acid (II-11) and nalidixic acid (Na salt) (II-12) were tested both as a free form
and a salt in order to test whether the aqueous solubility of chemicals might limit thepredictive power of the ROS assay. Photosensitivity was mentioned on both the US and

II-12

Nalidixic acid (Na salt)

the JPN label.
Norfloxacin was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1998a].

II-13

Norfloxacin

Photosensitivity wasmentioned on both the US and the JPN label.
Ofloxacin was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on

II-14

Ofloxacin

the 3T3 NRU-PT validation study [Spielmann et al, 1998a].

40
46

Photosensitivity wasmentioned on both the US and the JPN label.
Piroxicam was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1994a].
II-15

Piroxicam

Photosensitivity wasmentioned on both the US and the JPN label.
Promethazine HCl was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-16

Promethazine HCl

Photosensitivity

was mentioned on both the US and the JPN label.
Rosiglitazone was selected because 3T3 NRU-PT was positive, but high quality human

II-17

Rosiglitazone

data regarding phototoxicity was not available.
Tetracycline was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
the 3T3 NRU-PT validation study [Spielmann et al, 1994a].

II-18

Tetracycline

Photosensitivity wasmentioned on both the US and the JPN label.

Phototoxic non-drug chemicals
Anthracene was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on
II-19

Anthracene

the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
Avobenzone was reported with photoallergy negative by a photopatch test in the article
by Szczurko C et al. [1994] and Trevisi P et al. [1994], but was reported to induce
photoallergic reactions in the article by Schauder, S. et al. [1997].

Therefore,

avobenzone was classified as a phototoxic chemical because we were not able to
II-20

Avobenzone

completely judge that avobenzone was a non-phototoxic chemical.
Bithionol was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on

II-21

Bithionolthe 3T3 NRU-PT validation study [Spielmann et al, 1994a].
Hexachlorophene was selected from the list of positive chemicals used in the 3T3
NRU-PT validation study. Human data for this chemical was described as positive in thearticle on the 3T3 NRU-PT validation study [Masuda et al., 1971 and Spielmann et al,

II-22

Hexachlorophene

1998a].
Rose bengal was selected from the list of positive chemicals used in the 3T3 NRU-PT
validation study. Human data for this chemical was described as positive in the article on

II-23

Rose bengal

the 3T3 NRU-PT validation study [Spielmann et al, 1998a].

Non-phototoxic drugs

41
47

Aspirin was selected based on negative results of the 3T3 NRU-PT per the article byOnoue et al. [2010].
II-24

AspirinPhotosensitivity was no䡐 mentioned on either the US or the JPN

label.
Benzocaine was selected based on negative results of the 3T3 NRU-PT per the article byOnoue et al. [2010].

II-25

Benzocaine

Photosensitivity was no䡐 mentioned on either the US or the JPN

label.
Erythromycin was selected based on negative results of the 3T3 NRU-PT per the article
by Onoue et al. [2010].

II-26

Erythromycin

Photosensitivity was no䡐 mentioned on either the US or the

JPN label.
Penicillin G was selected from the list of negative chemicals used in the 3T3 NRU-PT
validation study [Spielmann et al, 1994a].

II-27

Penicillin G

Photosensitivity was no䡐 mentioned oneither the US or the JPN label.
Phenytoin was selected based on negative results of the 3T3 NRU-PT per the article byOnoue et al. [2010].

II-28

Phenytoin

Photosensitivity was no䡐 mentioned on either the US or the JPN

label.

Non-phototoxic non-drug chemicals
Bumetrizole was selected from UV absorbers. We thought that human data of this UV
absorber would be provided.

Human data for this chemical, however, was notavailable. Therefore after confirming negative result in 3T3 NRU-PT, this chemical
II-29

Bumetrizole

was classified as a non-phototoxic chemical.
Chemical No. II-30 was registered at first as benzylindene camphor sulphonic acid. This
was a UV absorber used in the 3T3 NRU-PT validation study. However, we carried out
the Phase 2 study without noticing a transcription error of the CAS number on the final
chemical list. Because we did not notice this mistake until after the Phase 2 study, thischemical was reclassified as a non-phototoxic chemical after confirming negative results

II-30

Camphor sulfonic acidin 3T3 NRU-PT and low molar extinction coefficient (MEC).
Chlorhexidine was selected from the list of negative chemicals used in the 3T3 NRU-PT
validation study [Spielmann et al, 1994a and 1998a]. Negative result in human was

II-31

Chlorhexidine

described in the article on the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
We originally intended to use cinnamic aldehyde, an aromatic ingredient used in
cinnamon, as Chemical No. II-32, because this chemical used in the 3T3 NRU-PT
validation study. However, we inadvertently added cinnamic acid instead of cinnamic
aldehyde to the list of chemicals.

Cinnamic acid is known to form a dimer by lightirradiation when in the solid state.

The difference between cinnamic aldehyde andcinnamic acid is that the former has a side chain of aldehyde and the later one of
II-32

Cinnamic acidcarboxyl.

In addition, we carried out 3T3NRU-PT for cinnamic acid and the result was

42
48

negative, just like cinnamic aldehyde.

Therefore, we concluded that either chemical

was suitable for the ROS assay validation study, because cinnamic acid resembles
cinnamic aldehyde structurally, and the result of 3T3 NRU-PT testing was the same.
Drometrizole was selected from UV absorbers. We thought that human data of this UV
absorber would be provided.

Human data for this chemical, however, was notavailable. Therefore after confirming negative result in 3T3 NRU-PT, this chemical
II-33

Drometrizole

was classified as a non-phototoxic chemical.
L-Histidine was selected from the list of negative chemicals used in the 3T3 NRU-PT

II-34

L-Histidine

validation study [Spielmann et al, 1994a].
Methylbenzylidene camphor was selected from UV absorbers.

This was in the list ofnegative chemicals in the 3T3 NRU-PT validation study and Human data for thisII-35

Methylbenzylidene

chemical was described as negative in the article on the 3T3 NRU-PT validation study

camphor[Spielmann et al, 1998b].
Octrizole was selected from UV absorbers. We thought that human data of this UV
absorber would be provided.

Human data for this chemical, however, was notavailable. Therefore after confirming negative result in 3T3 NRU-PT, this chemical
II-36

Octrizole

was classified as a non-phototoxic chemical.
Chemical No. II-37 was registered at first as octyl methoxycinnamate. This was UV
absorber used in the 3T3 NRU-PT validation study. However, we have carried out thePhase 2 study without noticing a transcribing error of the CAS number when we madethe final chemical list. Because we noticed to take it wrong after the Phase 2 study, thischemical was classified as non-phototoxic chemical again after confirming negative

II-37

Octyl methacrylate

result in 3T3 NRU-PT and low molar extinction coefficient (MEC).
Octyl methoxycinnamate was selected from UV absorbers.

This was in the list ofnegative chemicals in the 3T3 NRU-PT validation study and Human data for thischemical was described as negative in the article on the 3T3 NRU-PT validation study
II-38

Octyl methoxycinnamate[Spielmann et al, 1998b].
Octyl salicylate was selected from UV absorbers.

This was in the list of negative

chemicals in the 3T3 NRU-PT validation study and Human data for this chemical was
described as negative in the article on the 3T3 NRU-PT validation study [Spielmann etII-39

Octyl salicylateal, 1998b].
PABA was selected from the list of negative chemicals used in the 3T3 NRU-PT
validation study [Spielmann et al, 1994a and 1998a].

II-40

PABA

Negative result in animals was

described in the article on the 3T3 NRU-PT validation study [Spielmann et al, 1998a].
SDS was selected from the list of negative chemicals used in the 3T3 NRU-PT validation

II-41

SDS

study [Spielmann et al, 1994a].

43
49

UV-571 was selected from UV absorbers.
absorber would be provided.

We thought that human data of this UV

Human data for this chemical, however, was notavailable. Therefore after confirming negative result in 3T3 NRU-PT, this chemical
II-42

UV-571was classified as a non-phototoxic chemical.

Positive/Negative controls
Quinine HCl was used as positive control according to the method of Onoue et al.
[2008a].
PC

Quinine HCl

Quinine HCl was classified as a phototoxic chemical for human per the article

by Ljunggren et al [1986].

Photosensitivity was mentioned on the US label.

Sulizobenzone was used as negative control according to the method of Onoue et al.
[2008a].

Sulisobenzone was classified as a non-phototoxic chemical, because Humandata for this chemical was described as negative in the article on the 3T3 NRU-PT
NC

Sulisobenzone

validation study [Spielmann et al, 1998b].

44
50

Appendix 5 Test chemicals for Phase 2 study and code listin vivo

UV/vis absorptionb)

No.

Chemical name

CAS No.a)Laboratory

3T3
MEC

Ȝmax

(L/mol/cm)

(nm)

NRU

Animal

Human

1

2

3

Phototoxic drugs
II-1

Acridine

260-94-6

2773

354

+ 1)

+ 1)

+ 1)

C-130

B-090

A-005

II-2

Acridine HCl

17784-47-3

2635

354

+ 1)

+ 1)

+ 1)

C-126

B-086

A-001

II-3

Amiodarone HCl

19774-82-4

5400

290 c)

+ 2)

+ 2)

+ 2)

C-127

B-087

A-002

2)

2)

2)

C-106

B-066

A-026

II-4

Chlorpromazine HCl

69-09-0

1746

304

+

+

+

II-5

Doxycycline HCl

10592-13-93715

290 c)

+ 2)

+ 2)

+ 2)

C-116

B-076

A-036

II-6

Fenofibrate

49562-28-93514

290 c)

+ 1)

NA

+ 1)

C-139

B-054

A-014

+/- 1,3,4)

NA

+

1)

C-141

B-056

A-016

II-7

Furosemide

54-31-92650

290 c)

II-8

Ketoprofen

22071-15-4

2092

290 c)

+ 1)

- 1)

+ 1)

C-128

B-088

A-003

II-9

6-Methylcoumarine

92-48-83219

290 c)

+ 1)

+ 1)

+ 1)

C-113

B-073

A-033

II-10

8-MOP

298-81-7

3631

300

+ 2)

+ 2)

+ 2)

C-131

B-091

A-006

II-11

Nalidixic acid389-08-2

3192

331

+ 1)

+ 1)

+ 1)

C-137

B-052

A-012

II-12

Nalidixic acid (Na salt)

3374-05-8

3019

333

+ 1)

+ 1)

+ 1)

C-134

B-094

A-009

II-13

Norfloxacin70458-96-7

3562

323

+ 3)

+ 1)

+ 1)

C-110

B-070

A-030

II-14

Ofloxacin

82419-36-1

8443

290 c)

+ 1)

+ 1)

+ 1)

C-112

B-072

A-032

II-15

Piroxicam

36322-90-4

3304

352

- 2)

- 2)

+ 2)

C-135

B-095

A-010

II-16

Promethazine HCl

58-33-3

1558

297

+ 2)

NA

+ 2)

C-101

B-061

A-021

II-17

Rosiglitazone

122320-73-4

1765

311

+ d)

NA

NA

C-117

B-077

A-037

II-18

Tetracycline

60-54-83842

290 c)

+ 2)

+ 2)

+ 2)

C-102

B-062

A-022

+ 1)

+ 1)

C-121

B-081

A-041

7)

C-109

B-069

A-029

Phototoxic non-drug chemicals
II-19

Anthracene120-12-7

2315

355

+ 1)

II-20

Avobenzone

70356-09-1

7686

354

+

II-21

Bithionol

97-18-7

2462

321

+ 2)

+ 2)

+ 2)

C-115

B-075

A-035

1)

1)

+ 1,8)

C-107

B-067

A-027

5)

-

-

6)

+

II-22

Hexachlorophene70-30-4

2431

300

-

II-23

Rose bengal

632-69-9

19269

549

+ 1)

- 1)

+ 1)

C-104

B-064

A-024

Non-phototoxic drugs
II-24

Aspirin

50-78-2

80

290 c)

- 3)

NA

NA

C-140

B-055

A-015

II-25

Benzocaine

94-09-74273

290 c)

- 3)

NA

NA

C-114

B-074

A-034

-

3)

NA

NA

C-119

B-079

A-039

II-26

Erythromycin

114-07-80

290 c)

II-27

Penicillin G

113-98-40

290 c)

- 2)

NA

NA

C-118

B-078

A-038

0

290 c)

3)

NA

NA

C-145

B-060

A-020

II-28

Phenytoin

57-41-0-45
51

Appendix 5 Test chemicals for Phase 2 study and code list
(continued)
in vivo

UV/vis absorptionb)

No.

Chemical name

CAS No.a)Laboratory

3T3
MEC

Ȝmax

(L/mol/cm)(nm)

3896-11-5

3873

3144-16-9

NRU

Animal

Human

1

2

3

306

- d)

NA

NA

C-138

B-053

A-013

0

290 c)

- d)

NA

NA

C-132

B-092

A-007

Non-phototoxic non-drug chemicals
II-29
II-30

Bumetrizole
Camphor sulfonic
acid

II-31

Chlorhexidine

55-56-1

1338

290 c)

- 1)

NA

- 1)

C-144

B-059

A-019

II-32

Cinnamic acid140-10-3

3373

290 c)

- d)

NA

NA

C-123

B-083

A-043

II-33

Drometrizole

2440-22-4

3946

295

- d)

NA

NA

C-129

B-089

A-004

II-34

L-Histidine

71-00-1

0

290 c)

- 2)

NA

NA

C-111

B-071

A-031

36861-47-9

9200

304

- 9)

- 9)

- 9)

C-136

B-051

A-011

II-35

Methylbenzylidene
camphor

II-36

Octrizole3147-75-9

3958

296

- d)

NA

NA

C-133

B-093

A-008

II-37

Octyl methacrylate

688-84-6

0

290 c)

-

d)

NA

NA

C-105

B-065

A-025

5466-77-3

3000

290 c)

- 9)

- 9)

- 9)

C-142

B-057

A-017

II-38

Octyl
methoxycinnamate

II-39

Octyl salicylate

118-60-5

1500

290 c)

- 9)

- 9)

- 9)

C-120

B-080

A-040

II-40

PABA

150-13-02404

290 c)

- 2)

- 1)

NA

C-124

B-084

A-044

c)

2)

NA

NA

C-125

B-085

A-045

II-41

SDS

151-21-3

0

290

-

II-42

UV-571125304-04-3

1900

290 c)

- d)

NA

NA

C-122

B-082

A-042

Positive/Negative controls
PC

Quinine HCl

6119-47-7

1938

330

+ 3)

+ 10)

+ 11)

PC

PC

PC

NC

Sulisobenzone

4065-45-6

3519

290 c)

- 3)

NA

- 9)

NC

NC

NC

8-MOP: 8-methoxy psoralen, PABA: p-aminobenzoic acid, SDS: sodium dodecyl sulfate+ : Positive, - : Negative, +/- : Equivocal, NA : Not available, PC : Positive control, NC : Negative controla) CAS No.: Chemical abstracts service registry number, b) The UV/vis absorbance (290-700 nm) of most chemicals
was measured in 20 mM phosphate buffer (pH 7.4). However, the UV/vis absorbance of chemical Nos. II-19, II-20,
II-29, II-33 and II-36 were measured in methanol, because these chemicals were not solved in 20 mM phosphatebuffer (pH 7.4). In the each case, test chemicals were dissolved in DMSO at 10 mM and diluted with 20 mM
phosphate buffer (pH 7.4) or methanol. Final concentration of DMSO was unified to 0.5%. c) Ȝmax (nm) was a
wavelength at which the UV/vis absorbance shows a peak between 290 and 700 nm. Ȝmax (nm) was indicated as 290
nm in the case where the peak absorption is located below 290 nm and the maximum absorption is at 290 nm. d) In
vitro phototoxicity was assessed by the 3T3 NRU PT in the participating laboratories, according to the OECD 432
guideline.
1) Spielmann et al., 1998a, 2) Spielmann et al., 1994a, 3) Onoue et al., 2010, 4) Peters et al., 2002, 5) Gaspar et al.,2012, 6) ZEBET in house data, 7) Schauder et al., 1997, 8) Masuda et al., 1971, 9) Spielmann et al., 1998b, 10)Spielmann et al., 1994b, 11) Ljunggren et al., 1986

46
52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

Peer Review Panel Evaluation of the Reactive Oxygen Species (ROS)
Photosafety Assay

Japanese Center for the Validation of Alternative Methods
National Institute of Health Sciences
Tokyo, Japan
16 October 201371

Final Report
16 October 2013

Table of Contents
Executive Summary
Peer Review Panel Evaluation
1. Introduction
2. Regulatory Rationale
3. Scientific Rationale And Limitations
4. Test Method Protocol
5. Inter- And Intra-Laboratory Reproducibility
6. Validation Reference Chemicals
7. Test Method Predictivity
8. Availability Of Data
9. Data Quality
10. Applicability Domain
11. Proficiency Chemicals
12. Performance Standards
13. Advantages: Cost, Time, And Animal Welfare
14. Conclusions
Appendices1. Peer Review Panel Members
2. Acknowledgements
3. Glossary
4. References

272

Final Report
16 October 2013

Executive Summary
The Japanese Center for the Validation of Alternative Methods (JaCVAM) convened an
independent scientific peer review panel to evaluate the validation status of the Reactive
Oxygen Species (ROS) assay in accordance with established international criteria (OECD,2005). The ROS assay is a test method proposed as a component of an integrated photosafety
testing strategy to evaluate whether test substances such as pharmaceuticals have the potential
to cause phototoxicity.
The panel met initially in February and again in August 2013 in Tokyo, Japan. The panel
considered the reports of two international validation studies and a proposed ROS Assay
protocol at their initial meeting. The panel subsequently reviewed updated versions of the ROS
Assay protocol and the validation study reports as revised by the Validation Management Team
(VMT). This report summarizes the panel’s final evaluation and conclusions.
Overall conclusion: The panel concluded that the reproducibility and predictivity of the ROS
assay is sufficient to support its use in an integrated photosafety testing and decision strategy
for drug research and development. In this strategy, negative results in the ROS assay would
not require further testing in animals or other tests, while positive, weakly positive, andinconclusive results would proceed to the next level of testing in an in vitro test system such as
the 3T3 Phototoxicity Assay (OECD Test Guideline 432). The panel also concluded that use of
the ROS assay could potentially provide significant savings in time, cost and reduced animal
use for photosafety assessments. Furthermore, incorporating the ROS assay into a photosafety
testing strategy is expected to significantly reduce the overall number of substances that would
require additional testing in the in vitro 3T3 Phototoxicity Assay and subsequent testing inanimals.
Regulatory rationale: The panel concluded that the ROS Assay is applicable for use within the
ICH regulatory testing framework for photosafety evaluation of pharmaceutical products.
Regulatory authorities (e.g. PMDA/MHLW, U.S. FDA, EMEA) require non-clinical photosafety
testing prior to approving First-in-Human Phase I studies so that appropriate precautions andobservations can be taken during initial human studies. Such non-clinical photosafety testing
typically includes an assessment of the potential for a drug to cause phototoxic reactions, which
are characterized clinically by dermal redness, swelling, irritation, and inflammation. The panel
also agreed that the ROS assay is applicable to in-house drug research and development.
Scientific rationale: The panel recognized that ROS production is the most importantmechanism for chemically-induced phototoxicity, and is therefore a critical pathway initiating
event leading to phototoxicity. The ROS assay quantitatively measures two common reactive
oxygen species generated by photoreactive chemicals after exposure to simulated sunlight. In
this validation study, chemicals that did not produce sufficient ROS to meet the photoreactivity
threshold classification criteria for the ROS assay are uniformly non-phototoxic, while chemicals
that met or exceeded the photoreactivity classification criteria include all known phototoxicants.
Therefore if a chemical is not photoreactive in the ROS assay, it is unlikely that phototoxicity will
occur in living systems.
Limitations: The panel noted that the ROS assay assesses chemical photoreactivity in a nonbiological system, and therefore may overpredict phototoxicity potential since it does not assessthe direct interaction of chemicals with biological tissues. The assay may also overestimate the373Final Report
16 October 2013
potential for phototoxicity because some chemicals may not achieve sufficient concentration inskin for phototoxic reactions to occur, or photodegradation may occur. Accordingly, positive
results in the ROS assay are generally recommended for further evaluation in a photosafety
testing strategy.
Validation study reference chemicals: The panel agreed that the reference chemicals
selected for the validation studies were appropriate and sufficiently representative of thechemicals likely to be evaluated in the assay. The 42 reference chemicals incorporated most
known human phototoxicants and included 23 known positives and 19 negatives. The chemicals
were backed by data from human patch testing and in vitro 3T3 phototoxicity assay results. Alldata from the validation studies were made available in the validation study reports.
Assay Reproducibility: The panel concluded that the assay had excellent reproducibility bothwithin and between laboratories for the 42 reference chemicals evaluated in the validation
studies. Additionally, the positive and negative control chemicals had 100% reproducibility within
and between laboratories based on classification outcome, which further supports the
reproducibility of the ROS assay.
Test method predictivity: After reviewing analyses provided in the validation study reports, thepanel agreed that conducting a single assay per chemical provided optimal predictivity. The
panel concluded that the classification criteria for test outcomes have been appropriately
optimized to avoid false negatives while minimizing false positives. The panel also noted thatchemicals positive for both reactive oxygen species were uniformly phototoxic.
Data quality: The panel agreed that the high level of within and between laboratory
reproducibility suggested a consistently high level of quality of the validation studies. While thestudies were not conducted in strict accordance with GLPs, most of the labs were GLP certified.
The validation management team also confirmed that quality control audits found that validation
report data accurately reflected the raw data results.
Test method protocols: The panel considered the test method protocols used for the twovalidation studies and key aspects of a proposed standardized ROS assay protocol. The panel
recommended that the solar simulator should be equipped with an appropriate temperature
control unit or fan since ROS production can be influenced by temperature. The panel
concluded that the list of proficiency chemicals provided in the test method protocol forlaboratories to use to demonstrate ability to perform the assay was appropriate. The panel
recommended that each lab should develop historical positive and negative control value
acceptance ranges that can be used to determine the acceptability of an individual test. The
panel also agreed with the appropriateness of the reference chemicals identified for qualificationof solar simulators other than the two used in the validation studies.
Applicability domain: The applicability domain of the ROS assay is currently restricted to only
those chemicals that meet the solubility criteria outlined in the protocol. The panel
recommended that as experience is gained from use of the ROS assay, the applicability domain
could be more fully described in terms of physicochemical properties and/or chemical classes.
This will contribute to increased efficiency by providing criteria that can be used to identify
whether a chemical may be satisfactorily tested in the ROS assay, or whether an alternate
assay should be used initially.

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Peer Review Panel Evaluation of the ROS Assay
Introduction
The Japanese Center for the Validation of Alternative Methods (JaCVAM) convened an
independent scientific peer review panel to evaluate the validation status of the Reactive
Oxygen Species (ROS) Assay in accordance with established international validation andacceptance criteria (OECD, 2005). The ROS Assay is a test method proposed to evaluate
whether test substances such as pharmaceuticals may have the potential to cause
phototoxicity.
The panel met initially in February and again in August 2013 in Tokyo. The panel considered thereports of two international validation studies and a proposed outline for a ROS assay protocol
at their initial meeting. Following provision of a complete ROS assay protocol by the Validation
Management Team (VMT) and updating of the validation study reports, the panel met a second
time to complete its evaluation. In conducting its evaluation, the panel addressed each of theevaluation criteria that correspond to internationally harmonized validation and acceptancecriteria. This report summarizes the panel’s final evaluation and conclusions.
Evaluation Criterion 1: A rationale for the test method should be available, including
description of toxicological mechanisms, a clear statement of scientific need, andregulatory application.
The panel concluded that the ROS assay is applicable for use within the ICH regulatory testing
framework for photosafety evaluation of pharmaceutical products. Regulatory authorities (e.g.
PMDA/MHLW, U.S. FDA, EMEA, KFDA) require non-clinical photosafety testing prior toapproving First-in-Human Phase I studies so that appropriate precautions and observations canbe taken during initial human studies. Such non-clinical photosafety testing typically includes anassessment of the potential for a drug to cause phototoxic reactions, which are characterized bydermal redness, swelling, irritation, and inflammation. The panel also recognized that the ROS
assay is applicable to in-house drug research and development. A proposed integrated
photosafety testing strategy incorporating the ROS assay is provided below as Figure 1.
Chemicals that exhibit the potential for phototoxicity should be identified and if appropriate,
eliminated in the early stages of drug discovery and development. Ideally, drugs should not be
phototoxic. However, some beneficial drugs that have phototoxicity potential may be
unavoidable, in which case it is important to ensure that there are appropriate precautions on
drug labels so that patients can avoid exposures to sunlight that could lead to adverse reactions.
ROS production is the most important mechanism for inducing chemical phototoxicity.
Physicochemical tests such as the ROS Assay enable the identification of ROS production bychemicals after exposure to UV and/or visible light.
.

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Figure 1
Proposed integrated photosafety testing strategy incorporating the ROS
Photosafety Assay (courtesy of Dr. Hosoi)

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Evaluation Criterion 2: The relationship between the test method endpoint(s) and thebiological effect and to the toxicity of interest should be addressed, describing
limitations of the test methods.
Scientific rationale The ROS assay is based on identifying reactive oxygen species producedby photoreactive chemicals after exposure to UV and/or visible light. This mechanism is thebasis for phototoxic reactions in the skin of humans, e.g., redness, swelling, irritation, andinflammation. The panel recognized that ROS production is the most important mechanism forchemically-induced phototoxicity, and is therefore a critical pathway initiating event leading tophototoxicity. The ROS assay quantitatively measures two common reactive oxygen species
generated by photoreactive chemicals after exposure to simulated sunlight. In this validation
study, chemicals that did not produce sufficient ROS to meet the positive photoreactivity
threshold classification criteria for this assay are uniformly non-phototoxic, while chemicals that
met or exceeded the positive classification criteria include all known phototoxicants. Therefore if
a chemical is negative in the ROS assay it is unlikely that phototoxicity will occur in living
systems.
Limitations: The panel noted that the ROS assay assesses chemical photoreactivity in a nonbiological system, and therefore may overpredict phototoxicity potential since it does not assessthe direct interaction of chemicals with biological tissues. Additionally, the initiation of phototoxic
reaction in humans depends on pharmacokinetics and sufficient concentration in the target
tissue, which cannot be assessed in this assay. The assay may also overestimate the potential
for phototoxicity because some chemicals may not achieve sufficient concentration in skin forphototoxic reactions to occur, or photodegradation may occur. Accordingly, positive results in
the ROS assay are generally recommended for further evaluation in a photosafety testing
strategy.
Evaluation Criterion 3: A detailed test method protocol should be available.
The panel considered the test method protocols used for the two validation studies and key
aspects of a proposed standardized ROS assay protocol. The panel concluded that the
proposed ROS assay protocol was sufficiently detailed to allow for users to successfully performthe procedure. The panel also concluded that the protocol included adequate and appropriate
analysis and classification criteria. The panel recommended that the solar simulator should beequipped with an appropriate temperature control unit or fan since ROS production can be
influenced by temperature. The panel concluded that the list of proficiency chemicals provided
in the test method protocol for laboratories to use to demonstrate ability to perform the assay
was appropriate. The panel recommended that each lab should develop historical positive andnegative control value acceptance ranges that can be used to determine the acceptability of anindividual test.

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Evaluation Criterion 4: Within- and between-laboratory reproducibility of the test methodshould be demonstrated.
The panel concluded that the assay demonstrated excellent reproducibility both within andbetween laboratories for the 42 reference chemicals evaluated in the validation studies.
Additionally, the positive and negative control chemicals had 100% reproducibility within andbetween laboratories based on classification outcome, which further supports the reproducibilityof the ROS assay.
Evaluation Criterion 5: Demonstration of the test method’s performance should be basedon testing of representative, preferably coded reference chemicals.
The panel agreed that the reference chemicals selected for the validation studies wereappropriate, and sufficiently representative of the chemicals likely to be evaluated in the assay.
The 42 reference chemicals incorporated most known human phototoxicants and included 23
known positives and 19 negatives. The chemicals were backed by data from human patch
testing and in vitro 3T3 phototoxicity assay results. The validation reference chemicals wereappropriately coded to minimize bias by performing labs. All data from the validation studies
were made available in the validation study reports.
The panel noted the potential importance of chemical structure, and acknowledged the VMT for
incorporating chemical structures for all chemicals in the validation report. In addition, the panel
noted that the VMT also assessed and described whether the current drug label information forJapan and U.S. included precautionary language for phototoxicity.
Evaluation Criterion 6: Accuracy or predictive capacity should be demonstrated using
representative chemicals. The performance of test methods should have been evaluatedin relation to existing relevant toxicity data as well as information from the relevant target
species.
After reviewing analyses provided in the study reports, the panel agreed with the VMT that asingle assay per chemical provided optimal predictivity. The panel concluded that theclassification criteria for test outcomes had been appropriately optimized to avoid false
negatives while minimizing false positives (see ROS assay protocol judgment criteria).
Appropriate criteria are provided for photoreactive, weakly photoreactive, non-photoreactive,
and inconclusive classifications. In the first validation study (Atlas solar simulator), twophototoxic and one non-phototoxic reference chemicals were classified as inconclusive due tosolubility issues, and were not included in the integrated accuracy calculations. In the second
validation study (Seric solar simulator), three phototoxic and four non-phototoxic reference
chemicals were classified as inconclusive due to solubility issues, and were not included in the
integrated accuracy calculations.
All of the phototoxic reference chemicals that produced conclusive results were identified as
photoreactive in both validation studies, resulting in a sensitivity of 100% and a false negative
rate of 0%. In the first validation study, of the 18 non-phototoxic reference chemicals thatprovided conclusive results, 15 were identified as non-photoreactive and three were classified
as weakly photoreactive, resulting in a specificity of 83.3 %(15/18), and a false positive rate of
16.7% (3/18). In the second study, of the 15 non-phototoxic chemicals for which there wereconclusive results, 12 were identified as non-phototoxic, two were classified as weakly
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photoreactive, and one was classified as phototoxic, resulting in a specificity of 80% (12/15),
and a false positive rate of 20% (3/15). However, it is important to note that of the nonphototoxic chemicals producing photoreactive results, all three responses were categorized as
weakly photoreactive in the first study, and 2 of the 3 responses were categorized as weakly
photoreactive in the second study.
Evaluation Criterion 7: All data supporting the assessment of the validity of the test
method should be available for expert review.
All raw data for the two validation studies was provided in the validation study reports, which arereadily available electronically from the Japanese Center for the Validation of Alternative
Methods at the National Institute of Health Sciences, Tokyo, Japan.
Evaluation Criterion 8: Ideally, all data supporting the validity of a test method shouldhave been obtained in accordance with the principles of Good Laboratory Practice (GLP).
The panel concluded that there was a high level of within and between laboratory
reproducibility, which suggested a consistently high level of quality of the validation studies.
While the studies were not conducted in strict accordance with GLPs, six of the sevenlaboratories participating in the validation studies were GLP certified. This included two of threeof the labs in Study #1 (Atlas), and all four of the labs participating in Study #2 (Seric). There
was no significant variability between laboratories, which suggested a consistent level of quality.
The validation management team also confirmed that quality control audits found that validation
report data accurately reflected the raw data results.
.
Evaluation Criterion 9: The applicability domain of the validity of the test method should
be defined for expert review.
The applicability domain of the ROS Assay is currently restricted to only those chemicals thatmeet the solubility criteria outlined in the protocol. The panel recommended that as experienceis gained from use of the ROS assay, the applicability domain could be more fully described interms of physicochemical properties and/or chemical classes. This would contribute toincreased efficiency by providing criteria that can be used to identify whether a chemical may besatisfactorily tested in the ROS assay, or whether an alternate assay should be used initially.
Chemicals that are insoluble in the recommended vehicles and therefore are not suitable fortesting with this assay may be able to be tested in other vehicles, such as BSA, alcohol, andacetone. However, further characterization and standardization of procedures using thesealternative vehicles should be performed before incorporation into routine use.
Evaluation Criterion 10: Proficiency chemicals should be provided in the proposed
protocol.
The panel concluded that the list of 9 proficiency chemicals provided in the test method protocol
for laboratories to use to demonstrate ability to perform the assay was appropriate. These 9chemicals were selected from the validation study reference chemicals and represent a wide
range of responses in the assay as well as a wide range of solubilities.
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Evaluation Criterion 11: Performance standards should be developed for the proposed
protocol.
The panel agreed with the appropriateness of the 17 reference chemicals identified for
qualification of proposed solar simulators other than the two solar simulators used in thevalidation studies. The reference chemicals were appropriately selected from the reference
chemicals used for the validation studies. While performance standards were not specifically
proposed, the panel considered that these reference chemicals would be appropriate forincorporation in future performance standards for the ROS assay.

Evaluation Criterion 12: Are there advantages in terms of time, cost and animal welfare?
The ROS assay can potentially provide significant savings in time, cost and reduced animal use
when used in an integrated photosafety testing strategy by allowing decisions to be made earlier
and with fewer overall tests for many chemicals. These advantages are illustrated in Figure 1,
which shows that chemicals that are non-photoreactive in the ROS assay need not be tested inanimals or other tests. The ROS assay also reduces the number of chemicals which progress totesting in the 3T3 Phototoxicity Assay, with a subsequent reduction in the number of positive
results in the 3T3 assay that may progress to in vivo tests for confirmation.
Conclusion
The panel concluded that the reproducibility and predictivity of the ROS assay is sufficient tosupport its use in an integrated photosafety testing and decision strategy for drug research anddevelopment. In this integrated strategy, negative results in the ROS assay would not require
further testing in animals or other tests, while positive, weakly positive, and inconclusive results
would proceed to the next level of testing in an in vitro test system such as the 3T3 Phototoxicity
Assay (OECD Test Guideline 432). The panel also concluded that use of the ROS assay will
provide significant potential savings in time, cost and reduced animal use for photosafetyassessments. Furthermore, incorporating the ROS assay into a photosafety testing strategy will
significantly reduce the overall number of substances that require additional testing in the invitro 3T3 Phototoxicity Assay, and substantially reduce the number of substances that require
subsequent testing in animals.

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Appendix 1
Independent Peer Review Panel Members
Horst Spielmann, M.D., Panel Chairman
Dr. Spielmann is a Professor for Regulatory Toxicology in the Institute for Pharmacy, Faculty ofBiology, Chemistry, and Pharmacy at the Freie Universität Berlin in Berlin, Germany. He is theformer and first head of the National German Center for the Validation of Alternative Methods(ZEBET) at the Federal Institute for Risk Assessment (BfR) in Berlin, Germany, where hedirected the standardization and international validation of several in vitro methods and also
served as the head of the Department of Scientific Services. He served as Germany’s firstnational representative on the Scientific Advisory Committee for the European Centre for the
Validation of Alternative Methods at the European Commission’s Joint Research Centre in
Ispra, Italy, and has served as an expert for many years for the European Commission’s
Framework Programs on alternatives to animal testing and in vitro toxicology. In December
2012, he was appointed as the Animal Welfare Officer for the State of Berlin.
William S. Stokes, D.V.M., Rapporteur
Dr. Stokes is an Adjunct Professor in the Department of Molecular Biomedical Sciences,
College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, U.S.A.
He recently retired from the United States Public Health Service where he was an Assistant U.S.
Surgeon General. He served as the first Director of the U.S. National Toxicology Program’s
Interagency Center for the Evaluation of Alternative Toxicological Methods at the National
Institute of Environmental Health Sciences from 1997-2012, where he directed the international
validation and scientific peer review of numerous new test methods for regulatory safetyassessments. He also served as the first Executive Director of the U.S. Interagency
Coordinating Committee on the Validation of Alternative Methods from 1997-2012. He is a
Fellow of the Academy of Toxicological Sciences and a board certified environmental scientist,and is also board certified in laboratory animal medicine and animal welfare.
Ikuo Horii, Ph.D.
Dr. Horii is a global consultant for Pfizer and former Executive Director of Pfizer Drug Safety
Research and Development in Nagoya, Japan. He is a Certified Toxicologist in the JapaneseSociety of Toxicology with a background in biochemistry, pharmacology, pathology, andmolecular toxicology. He is currently a Visiting Professor at several institutions, including the
School of Pharmacy at Showa University in Japan, Dalian Medical University in China, andCambridge University in the United Kingdom. He is a Visiting Research Fellow at the National
Institute of Health Sciences in Tokyo, Japan and Lecturer at Kyoto University, Tokyo University,
and Chiba Institute of Science. He is President of Horii Science Associates and a board
member of the Japanese Society of Toxicology.
Bae-Hwan Kim, D.V.M., Ph.D.
Dr. Kim is a Professor in the Department of Public Health at Keimyung University in Daegu,
Republic of Korea, where he leads a biomedical research program and lectures in the Collegeof Natural Sciences. He previously worked in the pharmaceutical and cosmetic industry for 15
years as a Team Leader in the Preclinical Department. His research includes investigation of
the oxidative stress and oxidative photodamage induced by UV radiation and interventional
strategies for avoidance of UV irradiation damage. His focus is on the safety evaluation ofsubstances applied to the skin and the development of alternative methods to animal
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experiments. Dr. Kim serves on the Editorial Board of the Journal of Biomedical Research andis a Council Member of the Korean Association for Laboratory Animal Science and the Korean
Society for Alternatives to Animal Experiments.

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Appendix 2
Acknowledgements
The Peer Review Panel members gratefully acknowledge Steven Venti from BHK Limited for his
invaluable assistance with translation and editing during the peer review process. The Panelalso acknowledges the members of the Validation Management Team for the completeness ofthe validation study reports, their cooperation and responsiveness to requests for additional
information and analyses, and their responsive consideration of suggestions and updating of thevalidation study reports and test method protocol. Finally, the Panel expresses its appreciation
to Dr. Hajime Kojima and his staff at the National Institute of Health Sciences for their excellent
support and arrangements for the peer review panel meetings.

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Appendix 3

Glossary1

3T3 NRU-PT: In vitro 3T3 neutral red uptake phototoxicity test.
Dose of light: The quantity [= intensity Å~ time (seconds)] of UV or visible light incident on asurface, expressed in J/m2 or J/cm2.
Irradiance: The intensity of UV or visible light incident on a surface, measured in W/m2 or
mW/cm2.
MEC: Molar Extinction Coefficient (also called molar absorptivity) is a constant for any given
molecule under a specific set of conditions (e.g., solvent, temperature, and wavelength) andreflects the efficiency with which a molecule can absorb a photon (typically expressed as L mol1 cm-1).
Photoreactivity: the property of a chemical to react with another molecule as a consequence ofphoton absorption. Excitation of molecules by light can lead to generation of reactive oxygen
species (ROS) such as superoxide anion (SA) and singlet oxygen (SO) through energy transfer
mechanisms.
Phototoxicity: acute toxic response that is elicited after the first exposure of skin to certain
chemicals and subsequent exposure to light, or that is induced similarly by skin irradiation after
systemic administration of a chemical.
ROS: Reactive Oxygen Species, including superoxide anion (SA) and singlet oxygen (SO).
UVA: Ultraviolet light A (wavelengths between 320 and 400 nm).
UVB: Ultraviolet light B (wavelengths between 290 and 320 nm).
UVC: Ultraviolet light C (wavelengths between 190 and 290 nm).

1

Note: definitions derived from OECD TG 432 and the ROS assay protocol
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Appendix 4
References
1. OECD. Guidance Document No. 34: Guidance Document on the Validation andInternational Acceptance of New or Updated Test Methods for Hazard Assessment.
Organization for Economic Cooperation and Development, Paris, France. 2005.
Available at:
http://search.oecd.org/officialdocuments/displaydocumentpdf/?doclanguage=en&c
ote=env/jm/mono(2005)14

2. OECD. Test Guideline 432: In Vitro 3T3 NRU Phototoxicity Assay. OECD
Guidelines for the Testing of Chemicals, Section 4: Health Effects. Organization for
Economic Cooperation and Development, Paris, France. 2005. Available at:
http://www.oecd-ilibrary.org/environment/test-no-432-in-vitro-3t3-nru-phototoxicitytest_9789264071162-en

3. ROS Assay Validation Management Team. Validation report for the international
validation study on ROS (Reactive Oxygen Species) assay as a test evaluating
phototoxic potential of chemicals (Atlas Suntest version), 20 September 2013.

4. ROS Assay Validation Management Team. Validation report for the international
validation study on ROS (Reactive Oxygen Species) assay as a test evaluating
phototoxic potential of chemicals (Seric version), 9 October 2013.

5. ROS Assay Validation Management Team. Reactive Oxygen Species (ROS)
Assay to Examine Photoreactivity of Chemicals: ROS Assay Protocol, version 3.1,
20 September 2013.

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REACTIVE OXYGEN SPECIES (ROS) ASSAY TO EXAMINEPHOTOREACTIVITY OF CHEMICALS

Issued by: ROS assay Validation Management Team
Date: 28 November 2014.

1.

INTRODUCTION

The purpose of this document is to recommend a protocol for assaying the photoreactivity ofchemicals based on reactive oxygen species (ROS). Photoreactivity is defined as the property of achemical to react with another molecule as a consequence of photon absorption.

Excitation ofmolecules by light can lead to generation of ROS such as superoxide anion (SA) and singlet oxygen(SO) through energy transfer mechanisms.

The ROS assay does not measure phototoxicity directly,

but rather is a physicochemical test that can be applied to that purpose, similar to measurement of
UV absorbance.

2.

INITIAL CONSIDERATION

Validation studies conducted by JaCVAM showed that the ROS assay has 100% sensitivity forpredicting phototoxicants but can result in some false positives [1-3].

Based on the results of thevalidation studies, conducting this assay would classify a test chemical into one of the following:
photoreactive, weakly photoreactive, non-photoreactive, or inconclusive.

Photoreactive, weakly

photoreactive, or inconclusive results would be a flag for follow-up phototoxicity assessment.
Non-photoreactive results indicate a very low probability of phototoxicity, and no further
phototoxicity testing would be suggested.

In the ROS assay, measurement is first made at aconcentration of 200 μM (final concentration).

If interference such as precipitation or coloration(exhibiting peak absorbance at 440 or 560 nm) is observed at 200 μM, measurements are made at 20
μM.

When precipitation or coloration is found at 20 μM, the substance is considered incompatible

with the ROS assay.

Since the ROS assay is designed to evaluate directly the photoreactivity ofchemicals, it is not suitable for detecting chemicals that induce in vivo phototoxicity by indirect
mechanisms such as porphyria and pseudoporphyria.

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PRINCIPLE OF THE TEST METHOD

3.

Drug-induced photoirritation can be defined as an inflammatory reaction of the skin after topical orsystemic administration of pharmaceutical substances.

There are several classes of drugs(including antibacterials, thiazide diuretics, non-steroidal anti-inflammatory drugs, quinolones, andtricyclic antidepressants) which are nontoxic by themselves but could become reactive when
exposed to environmental light and thereby result in undesired side effects.

The primary event inany photosensitization process is the absorption of photons of a wavelength that induces excitationof the chromophore.

The excitation energy is often transferred to oxygen molecules, followed bygeneration of ROS, including SA through type I reaction and SO through type II reaction byphoto-excited drug molecules.
phototoxic response.

These appear to be the principal intermediate species in the

From the standpoint of risk assessment, previous research has demonstrated

that determination of ROS from pharmaceutical substances irradiated with UVA/B and visible lightwould be of help in recognizing their phototoxic potential.
In the ROS assay, SO generation was detected by spectrophotometric measurement of
p-nitrosodimethyl aniline (RNO) bleaching, followed by decreased absorbance of RNO at 440 nm[4].

Although SO does not react chemically with RNO, the RNO bleaching is a consequence of SO

capture by the imidazole ring, which results in the formation of a trans-annular peroxideintermediate capable of inducing the bleaching of RNO, as follows:
SO+ Imidazole ĺ [Peroxide intermediate] ĺ Oxidized imidazole[Peroxide intermediate] + RNO ĺ RNO + Products
SA generation was detected by the observing the reduction of nitroblue tetrazolium (NBT) as
indicated below; NBT can be reduced by SA via a one-electron transfer reaction, yielding partiallyreduced (2 e-) monoformazan (NBT+) as a stable intermediate [5].

Thus, SA can reduce NBT to

NBT+, the formation of which can be monitored spectrophotometrically at 560 nm.
SA + NBT ĺ O2 + NBT+4.

DESCRIPTION OF THE TEST METHODTechnical equipment
Solar simulators:
Suntest CP series (Atlas Material Technology, Chicago, IL, USA) or SXL-2500V2 (Seric, Tokyo,
Japan) with a fan and UVC cut filter (Spectrum shown in Appendix 1)
UVA detector: e.g. #0037 (Dr. Hönle, München, German) or UD series (Topcon, Tokyo, Japan)
Quartz reaction container (Ozawa Science, Aichi, Japan, Appendix 2) or equivalent
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Microplate spectrophotometer, equipped with 440 and 560 nm filters
Microscope
Thermometer
Vortex mixer
Plate shaker
Sonicator
Pipettes
Polypropylene tubes
Plastic 96-well plates (clear, non-treated, flat-bottom)
Plastic and glass laboratory ware

Solar simulator
An appropriate solar simulator is to be used for irradiation of UV and visible light. The irradiation
power distribution is to be kept as close to that of outdoor daylight as possible by using anappropriate UVC cut filter.

Recommended solar simulators and UVA intensity on the plate position

measured by UVA detector #0037 (Dr. Hönle) are as follows:
Suntest CPS+ or CPS (Atlas) with UV cut filter (<290 nm),
1.8 to 2.2 mW/cm2 (e.g. the indicator setting value of 250 W/m2 for CPS+) for 1 hour,
6.5 to 7.9 J/cm2 of UVA intensity (Appendix 1)
SXL-2500V2 (Seric) with UV cut filter (<300 nm),
3.0 to 5.0 mW/cm2 for 1 hour,
11 to 18 J/cm2 of UVA intensity (Appendix 1)
The solar simulator is to be equipped with an appropriate temperature control or fan to stabilize thetemperature during irradiation, because ROS production is affected by temperature.
temperature for a solar simulator with temperature control is 25°C.
range during irradiation is 20° to 29°C.

Standard

The acceptable temperature

If a solar simulator other than the two recommended

models is used, the reference chemical set listed in section 6 is to be tested prior to performing the
ROS assay to ensure that measured values of SO and SA are close to those mentioned in section 6.

Quartz reaction container
A quartz reaction container is used to avoid loss of UV due to passing through a plastic lid andvaporization of the reaction mixture [6].

A made-to-order container (See Appendix 2.) or its

equivalent is recommended.

If a different container is used, a lid or seal with high UVtransmittance must be used.

In this case, a feasibility study using the reference chemicals is to be

conducted to determine an appropriate level of exposure to UV and visible light.

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Reagents
The following reagents are to be used and stored according to the instructions of manufacturers.
NaH2PO4࣭2H2O (CAS No. 13472-35-0)
Na2HPO4࣭12H2O (CAS No. 10039-32-4)
p-Nitrosodimethylaniline (RNO, CAS No. 138-89-6)
Imidazole (CAS No. 288-32-4)
Nitroblue tetrazolium chloride (NBT, CAS No. 298-83-9)
Purified waterPreparation of reagents
All reagents are to be sonicated and used within 1 month after preparation.

Representative

preparation methods are shown as follows.
20 mM sodium phosphate buffer (NaPB), pH 7.4
Weigh 593 mg of NaH2PO4࣭2H2O and 5.8 g of Na2HPO4࣭12H2O, add 900 mL of purified
water, adjust with HCl to a pH of 7.4, dilute with purified water up to 1 L, and mix.
Store in a refrigerator or at room temperature.
0.2 mM p-Nitrosodimethylaniline (RNO)
Dissolve 3 mg of RNO in 100 mL of 20 mM NaPB.
Store in a refrigerator and protect from light.
20 mM imidazole
Dissolve 13.6 mg of imidazole in 10 mL of 20 mM NaPB.
Dilute the 2×10-2 M imidazole solution 100 times with 20 mM NaPB.
Store in a refrigerator and protect from light.
0.4 mM nitroblue tetrazolium chloride (NBT)
Dissolve 32.7 mg of NBT in 100 mL of 20 mM NaPB.
Store in a refrigerator and protect from light.

Test chemicals
Test chemicals are to be stored appropriately until termination of the study and their stability during
the test period is to be confirmed.
used.

One concentration level, 200 μM (final concentration), is to be

A 20-μM concentration can be used if precipitation before light exposure, coloration, orother interference is observed in the reaction mixture at 200 μM.

Preparation of test chemical solutions
The test chemical solutions are to be prepared using a solvent just before use.
to be weighed in a tube, and solvent added to a concentration 10 mM.
490Each test chemical is

The tube is to be mixed with

ROS Assay Protocol
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28November 2014a vortex mixer and sonicated for 5 to 10 minutes under UV-cut illumination or shade.
preparations are to be protected from light.
200 μM.

All

The final concentration in a reaction mixture is to be

When precipitation before light exposure, coloration, or other interference is observed in

the reaction mixture at 200 μM, a 1-mM solution (20 μM as the final concentration) is to beprepared using the solvent.

For chemicals that are not soluble in DMSO, 20 μL of DMSO is to becontained in the reaction mixture.

Positive and negative control chemicals
Quinine hydrochloride (CAS No. 6119-47-7) is to be used at 200 μM (final concentration) as apositive control.

Sulisobenzone (CAS No. 4065-45-6) is to be used at 200 μM (final concentration)

as a negative control.

Preparation of positive and negative control chemical solutions
Stock solutions of quinine and sulisobenzone are to be prepared at 10 mM each in DMSO (final
concentration of 200 μM) according to the above procedure, divided into tubes, and stored in afreezer (generally below -20ÛC) for up to 1 month. The stock solution is to be thawed just before
the experiment and used within the day.

Solvent
Use analytical grade DMSO at first.

For chemicals that are not soluble in DMSO, 20 mM of NaPB

is to be used. When a test chemical is insoluble in either DMSO or 20 mM NaPB, try bovine serum
albumin (BSA) or other solvent [7-8]. Prior to use of BSA or other solvent, however, perform afeasibility study (see Section 6) using the reference chemicals to determine appropriate test
conditions. The results of ROS assays using BSA or other solvents, however, are not suitable forregulatory purposes until these solvents have been properly evaluated.

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Test procedure
A tube (e.g. 1.5 mL micro tube) and a plastic clear flat bottomed 96-well microplate are to be used.
The reaction mixture is to be prepared by vortex mixing and/or sonication under UV-cut illuminationor shade.

The same volume of DMSO, 20 ȝL, is to be added in a blank instead of test chemicalsolution.
SO

SA

20 mM NaPB

480 ȝL

20 mM NaPB

855 ȝL

Imidazole

250 ȝLNBT

125 ȝLRNO

250 ȝL

Chemical

20 ȝLChemical20 ȝLMix (Vortex and sonication for 5 to 10 min)Add 200 ȝL of mixture to each well (n=3)1
Check solubility using a microscope at 100×2 and coloration3
Pre-read Abs at 440 and 560 nm after shaking for 5 sec
Light exposure for 1 hr4
Read A440 and A560 after shaking for 1 min and check coloration3
Notes
1) Avoid using peripheral wells. More than one test chemical can be tested on a plate. A typical
96-well plate configuration is as follows:

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2) Some chemicals might precipitate in the reaction mixture.
solubility prior to irradiation.

It is therefore important to check

Solubility of each reaction mixture in its well is to be observedwith a microscope prior to irradiation. Test chemical concentrations are to be selected so as
to avoid precipitation or cloudy solutions.
3) The reaction mixture is to be checked for coloration with the naked eye.
4) The 96-well plate is to be placed in the quartz reaction container. A quartz cover is to be seton the plate and fastened with bolts.

Ensure that temperature and other ambient conditions

are stable when using the solar simulator. Measure UVA intensity and temperature at theplate position using a UVA detector and thermometer both before and after irradiation.

5.

DATA AND REPORTING

Data analysis
Data from three wells for each chemical concentration is used to calculate mean and standard
deviation.
SO
Decrease of A440 ×1000 = [A440 (–) – A440 (+) – (a – b)] ×1000
A440 (–): Absorbance before light exposure at 440 nm
A440 (+): Absorbance after light exposure at 440 nm
a: Blank before light exposure (mean)b: Blank after exposure (mean)
SA
Increase of A560 ×1000 = [A560 (+) – A560 (–) – (b – a)] ×1000
A560 (–): Absorbance before light exposure at 560 nm
A560 (+): Absorbance after light exposure at 560 nm
a: Blank before light exposure (mean)b: Blank after exposure (mean)

Criteria for data acceptance
The following criteria are to be satisfied in each experiment.
No precipitation of test chemical in the reaction mixture before light exposure.
No coloration of test chemical in the reaction mixture before or after light exposure.
No technical problems, including prescribed temperature range, when collecting data set.
Raw OD value: 0.02 to 1.5
Historical positive and negative control values are to be developed by each laboratory based on a
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mean +/-2 SD.

The following range was defined based on the 95% confidence interval (mean+/- 1.96SD) obtained from the validation data.

When a solar simulator other than arecommended model is used, establish modified criteria based on 95% confidence interval.
Positive control value at 200 ȝM (mean of 3 wells)
SO: 319 to 583
SA: 193 to 385
Negative control value at 200 ȝM (mean of 3 wells)
SO: -9 to 11
SA: -20 to 2

Criteria for judgment
Each test chemical is to be judged as follows:
Judgment1, 2Concentration3

SO (mean of 3 wells)

Photoreactive

200 ȝM

25

and

70

<25 and/or I4

and

70

25

and

<70 and/or I4

SA (mean of 3 wells)

Weakly photoreactive

200 ȝM<25

and20, <70

Non-photoreactive

200 ȝM

<25

and

<20

Inconclusive

The results do not meet the above-mentioned criterion.

Notes
1. A single experiment is sufficient for judging
Photoreactive

results, because the ROS assay shows good intraandinter-laboratory

reproducibility

in

70

the

validation studies.

SA

2. If precipitation, coloration, or other interference isobserved at both 20 and 200 ȝM, the chemical is

Weaklyphotoreactive
20
NonͲphotoreactive
25

considered incompatible with the ROS assay andSOjudged as inconclusive.

3. Twenty ȝM can be used for judgment when precipitation or coloration is observed at 200 ȝM.
For regulatory purposes, the stability of the test chemical in the reaction mixture both before andafter light exposure is to be confirmed when results at 20 ȝM are used for judgment as a
non-photoreactive chemical for which no further phototoxicity testing is necessary.
4. Interference such as precipitation or coloration.

Data quality
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Studies for regulatory purposes are to be conducted to the highest of quality standards, with datacollection records readily available, in compliance with GLP/GMP regulations whenever possible,
and all documents checked by the Quality Assurance Unit of the laboratory.

Test report
The test report must include the following information:
Test chemical
Name and lot No.
Physical nature and purity
Storage condition
Stability during the test period
UV/vis absorption spectrum, maximum molar extinction coefficient at 290 to 700 nm, and/or
photostability, if known
Preparation of test chemical solution
Final concentrations tested
Control chemicals
Name, manufacturer, and lot No.
Physical nature and purity
Storage condition
Preparation of control chemical solutions
Final concentrations tested
Solvent
Name, manufacturer, and lot No.
Justification for choice of solvent
Irradiation condition
Manufacturer and type of the solar simulator used
Rationale for selection of the solar simulator used
UVA detector used
UVA irradiance, expressed in mW/cm2
UVA dose, expressed in J/cm2
Temperature before and after irradiation
ROS assay procedure
Acceptance and decision criteria
Results
Discussion
Conclusions
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Archives
The study report and all raw data is to be retained according to the SOP in the testing facility.

6.

REFERENCE CHEMICALS FOR THE FEASIBILITY STUDY

To perform a ROS assay, it is necessary first to ensure irradiation conditions that satisfy therecommended criteria using the positive and negative controls, after which reference chemicals areto be tested in a feasibility study.

The reference chemical set, as selected from the validation

studies, and acceptable value ranges are shown in Table 1 and Table 2. It is important that thevalues for SO and SA obtained in the feasibility study be similar to the following values.
Table 1

Standard chemical set for laboratories to demonstrate proficiency using solar simulators ofSuntest CPS/CPS+ (Atlas) or SXL-2500V2 (Seric) and the acceptable range

No.

Chemical

CAS No.

SO

SA

Solvent

Concentration

11

Doxycyclinehydrochloride

10592-13-9

115 to 429

230 to 468

DMSO

200 μM

12

Norfloxacin70458-96-7

131 to 271

57 to 161

DMSO

200 μM

13

8-Methoxy
psoralen298-81-7

31 to 137

0 to 126

DMSO

200 μM

14

Fenofibrate

49562-28-9

77 to 203

-31 to 11

DMSO

20 μM

15

p-Aminobenzoicacid150-13-0-8 to 12

-11 to 7

DMSO

200 μM

16

Benzocaine

94-09-7-7 to 9-7 to 17DMSO

200 μM

17

Erythromycin

114-07-8-15 to 11-9 to 21DMSO

200 μM

18

Octyl salicylate

118-60-5-5 to 11-8 to 20

DMSO

20 μM

19

L-Histidine

71-00-1-8 to 128 to 120

NaPB

200 μM

The values were calculated as mean +/- 1.96 SD from the validation data.
Note: Underline parts were corrected because of errors in writing.

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Table 2

Recommended chemical set for the other solar simulators and the acceptable range

No.

Chemical

CAS No.

SO

SA

Solvent

Concentration

21

Acridine

260-94-6

182 to 328

121 to 243

DMSO

200 μM

22

Chlorpromazinehydrochloride

69-09-0-56 to 70

66 to 106DMSO

200 μM

23

Diclofenac

15307-79-6

34 to 416

47 to 437

DMSO

200 μM

24

Doxycyclinehydrochloride

10592-13-9

115 to 429

230 to 468

DMSO

200 μM

25

Furosemide

54-31-931 to 225

-7 to 109

DMSO

200 μM

26

Ketoprofen

22071-15-4

120 to 346

77 to 151

DMSO

200 μM27

8-Methoxy
psoralen298-81-7

31 to 137

0 to 126

DMSO

200 μM

28

Nalidixic acid389-08-2

54 to 246

88 to 470

DMSO

200 μM

29

Norfloxacin70458-96-7

131 to 271

57 to 161

DMSO

200 μM

30

Omeprazole

73590-58-6-221 to 103

30 to 216

DMSO

200 μM

31

Promethazinehydrochloride

58-33-3

20 to 168

-3 to 77

DMSO

200 μM

32

Fenofibrate

49562-28-9

77 to 203

-31 to 11

DMSO

20 μM

33

p-Aminobenzoicacid150-13-0-8 to 12

-11 to 7

DMSO

200 μM

34

Benzocaine

94-09-7-7 to 9-7 to 17DMSO

200 μM

35

Erythromycin

114-07-8-15 to 11-9 to 21DMSO

200 μM

36

Octyl salicylate

118-60-5-5 to 11-8 to 20

DMSO

20 μM

37

L-Histidine

71-00-1-8 to 128 to 120

NaPB

200 μM

The values were calculated as mean +/- 1.96 SD from the validation data.

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7.

GLOSSARY

ROS: Reactive Oxygen Species, including superoxide anion (SA) and singlet oxygen (SO).
3T3 NRU-PT: In vitro 3T3 neutral red uptake phototoxicity test.
Irradiance: The intensity of UV or visible light incident on a surface, measured in W/m2 or
mW/cm2.
Dose of light: The quantity [= intensity × time (seconds)] of UV or visible light incident on asurface, expressed in J/m2 or J/cm2.
MEC: Molar Extinction Coefficient (also called molar absorptivity) is a constant for any given
molecule under a specific set of conditions (e.g., solvent, temperature, and wavelength) and reflects
the efficiency with which a molecule can absorb a photon (typically expressed as L mol-1 cm-1).
Photoreactivity: The property of chemicals that react with another molecule as a consequence ofabsorption of photons.
Phototoxicity: An acute light-induced tissue response to a photoreactive chemical.
UVA: Ultraviolet light A (wavelengths between 320 and 400 nm).
UVB: Ultraviolet light B (wavelengths between 290 and 320 nm).
UVC: Ultraviolet light C (wavelengths between 190 and 290 nm).

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REFFERENCES[1] ROS assay validation management team. Validation report for the international validation study
on ROS (Reactive Oxygen Species) assay as a test evaluating phototoxic potential of chemicals(Atlas Suntest version), 2013.
[2] ROS assay validation management team. Validation report for the international validation study
on ROS (Reactive Oxygen Species) assay as a test evaluating phototoxic potential of chemicals(Seric version), 2013.
[3] Onoue S, Hosoi K, Wakuri S, Iwase Y, Yamamoto T, Matsuoka N, Nakamura K, Toda T,
Takagi H, Osaki N, Matsumoto Y, Kawakami S, Seto Y, Kato M, Yamada S, Ohno Y, Kojima
H. Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen
species assay for chemical photosafety evaluation. J Appl Toxicol, Article first published online,
13 June, 2012 (DOI: 10.1002/jat.2776).
[4] I. Kraljiü, S. El Mohsni. A new method for the detection of singlet oxygen in aqueous solutions.
Photochem Photobiol, 28, 577-81, 1978.
[5] Pathak MA, Joshi PC. Production of active oxygen species (1O2 and O2-.) by psoralens andultraviolet radiation (320-400 nm). Biochim Biophys Acta, 798, 115-26, 1984.
[6] Onoue S, Igarashi N, Yamada S, Tsuda Y. High-throughput reactive oxygen species (ROS)
assay: an enabling technology for screening the phototoxic potential of pharmaceuticalsubstances. J Pharm Biomed Anal, 46, 187-93, 2008.
[7] Onoue S, Yamauchi Y, Kojima T, Igarashi N, Tsuda Y. Analytical studies on photochemical
behavior of phototoxic substances; effect of detergent additives on singlet oxygen generation.
Pharm Res, 25, 861-868, 2008.
[8] Onoue S, Kato M, Yamada S. Development of an albuminous reactive oxygen species assay forphotosafety evaluation under experimental biomimetic conditions. J Appl Toxicol, Article firstpublished online, 28 Jan, 2013 (DOI: 10.1002/jat.2846).

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Appendix 1

Spectrum of solar simulators used in the validation studies

Suntest CPS/CPS+(Atlas)SXL-2500V2㻌
(Seric)Sun light

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Appendix 2

Quartz reaction container used in the validation studies
166 mm

86 mm

24 mm

14.5 mm

127 mm15
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Appendix 3

Amendment

Version 3.2
Page 10: The SA values of benzocaine and erythromycin were corrected in Table 1 because of errors
in writing.
Incorrect

Correct

Benzocaine

7 to 17-7 to 17

Erythromycin

9 to 21-9 to 2116102